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Abstract:

A metal nanoparticle-phosphopeptide complex comprising a metal
nanoparticle and a phosphopeptide is provided. The phosphopeptide
comprises two or more contiguous peptide motifs and two or more
phosphorus-containing groups capable of interacting with the surface of
the metal nanoparticle. The amino acids at the equivalent position in
each peptide motif have similar structural and/or electronic properties.
Each phosphorus-containing group is bound to an amino acid in the two or
more contiguous peptide motifs. Methods for preparing the metal
nanoparticle-phosphopeptide complex are also provided.

Claims:

1. A metal nanoparticle-phosphopeptide complex comprising: a metal
nanoparticle; and a phosphopeptide comprising two or more contiguous
peptide motifs and two or more phosphorus-containing groups capable of
interacting with the surface of the metal nanoparticle, wherein the amino
acids at the equivalent position in each peptide motif have similar
structural and/or electronic properties, and wherein each
phosphorus-containing group is bound to an amino acid in the two or more
contiguous peptide motifs.

2. The complex of claim 1, wherein the nanoparticle comprises one or more
metals selected from the metals in groups 3 to 12 of the periodic table.

3. The complex of claim 2, wherein the one or more metals are selected
from the metals in periods 4 to 6 of groups 8 to 11 of the periodic
table.

4. The complex of claim 1, wherein the metal nanoparticle is an iron,
ruthenium, palladium, or gold nanoparticle.

5. The complex of claim 1, wherein the phosphopeptide is adsorbed to the
surface of the metal nanoparticle.

6. The complex of claim 1, wherein the two or more phosphorus-containing
groups are bound to amino acids at the equivalent position in each
peptide motif.

7. The complex of claim 1, wherein each peptide motif is from 3 to 6
amino acids in length.

8. The complex of claim 7, wherein each peptide motif is 3 amino acids in
length.

9. The complex of claim 1, wherein the phosphopeptide is from 6 to 50
amino acids in length.

10. The complex of claim 1, wherein the phosphopeptide further comprises
one or more groups that mitigates aggregation of the metal
nanoparticle-phosphopeptide complex with metal nanoparticles or other
metal nanoparticle-phosphopeptide complexes.

11. The complex of claim 10, wherein the group that mitigates aggregation
is a charged peptide.

12. The complex of claim 1, wherein each amino acid of the two or more
peptide motifs is independently a natural amino acid; or an unnatural
amino acid residue of the formula (II): ##STR00041## wherein: R1
and R3 are each hydrogen; R2 is C1-6alkylheteroaryl; and m
is 0 and p is 0.

13. The complex of claim 12, wherein each phosphorus-containing group is
bound to the oxygen atom of a serine, threonine, or tyrosine residue
hydroxyl group; or the heteroaryl group of an amino acid residue of the
formula (II).

14. The complex of claim 13, wherein each phosphorus-containing group
bound to a natural amino acid is --P(O)(OH)2; and each
phosphorus-containing group bound to an unnatural amino acid is
C1-6alkylphosphonate.

15. A method for preparing a metal nanoparticle-phosphopeptide complex,
the method comprising contacting a metal compound; and a phosphopeptide
comprising two or more contiguous peptide motifs and two or more
phosphorus-containing groups capable of interacting with the surface of
the metal nanoparticle, wherein the amino acids at the equivalent
position in each peptide motif have similar structural and/or electronic
properties, and wherein each phosphorus-containing group is bound to an
amino acid in the two or more contiguous peptide motifs; in a liquid
reaction medium under conditions that form a metal
nanoparticle-phosphopeptide complex.

16. The method of claim 15, wherein the method comprises contacting the
metal compound and phosphopeptide with a reducing agent in the liquid
reaction medium.

17. The method of claim 16, wherein the metal compound is a metal salt
comprising a metal cation.

18. The method of claim 16, wherein the metal compound is a compound of a
metal selected from the metals in periods 4 to 6 of groups 8 to 11 of the
periodic table.

19. The method of claim 16, wherein the reducing agent is sodium
borohydride.

21. The method of claim 15, wherein the method comprises contacting the
metal compound and phosphopeptide under conditions that precipitate the
metal nanoparticle-phosphopeptide complex.

22. The method of claim 21, wherein the method comprises contacting the
metal compound and phosphopeptide with hydroxide or chalcogen anions.

23. The method of claim 21, wherein the method comprises contacting two
or more metal compounds.

24. The method of claim 21, wherein the metal compound is a compound of a
metal selected from the metals in groups 3 to 12 of the periodic table.

25. The method of claim 21, wherein the metal compound is an iron (II) or
iron (III) salt.

Description:

TECHNICAL FIELD

[0001] The present invention generally relates to metal nanoparticles. In
particular, the present invention relates to metal
nanoparticle-phosphopeptide complexes, methods for preparing metal
nanoparticles and metal nanoparticle-phosphopeptide complexes, and to
metal nanoparticles and metal nanoparticle-phosphopeptide complexes
prepared according to those methods. The present invention also relates
to phosphopeptides and to compositions comprising metal nanoparticles and
those phosphopeptides, and to compositions comprising metal
nanoparticle-phosphopeptide complexes, and kits. The present invention
also relates to uses of the metal nanoparticles and metal
nanoparticle-phosphopeptide complexes in the manufacture of medicaments,
in methods of treatment and imaging, and as catalysts.

BACKGROUND OF THE INVENTION

[0002] Metallic nanoparticles exhibit unusual optical, thermal, chemical
and physical properties, due to the large proportion of high-energy
surface atoms compared to bulk solid and to the nanometer-scale mean free
path of electrons in the metal (˜10-100 nm for many metals at room
temperature.

[0003] Some metal nanoparticles have significant potential as catalysts
that, due to the ability to lower the activation energy of certain
reactions, facilitate the synthesis of important chemicals. Many
transition metals in their bulk state already possess catalytic
properties. Nanoparticles of such metals can have significantly greater
catalytic activities, due to the large specific surface areas of the
nanoparticles, which may open further fields of application.

[0004] Metal nanoparticles with increased the catalytic activity, relative
to bulk metal, allow the amount of the metal used to be reduced while
preserving the same level of catalyst performance. This can provide
significant cost benefits.

[0005] Metal nanoparticles may also have useful magnetic properties.
Magnetic nanoparticles are currently used as magnetic media storage, but
are also of great interest for their potential applications in medicine.
Potential medicinal applications include cancer treatment by
hyperthermia, contrast enhancement in medical imaging, new drug delivery
methods, etc.

[0006] Iron nanoparticles are of particular interest. Iron nanoparticles
exhibit strong ferromagnetic or ferrimagnetic behaviour, and
super-paramagnetic properties when the particle size is less than about
10 nm. As a result, iron is the most widely used metal for the
preparation of magnetic nanoparticles and their applications.

[0007] Many methods are available for the synthesis of metal
nanoparticles: thermal or sonochemical decomposition, hydrothermal
synthesis, vapor phase synthesis, laser pyrolysis, etc. However, these
methods typically require the use of complex equipment, high
temperatures, high pressures, and/or harsh organic solvents.

[0008] Wet chemical techniques involving, for example, the reduction of
metal salts have proven to be particularly efficient and can be performed
in water without any special equipment (see, for example, K. J. Carroll
et al., J. Appl. Phys., 2010, 107 and R. Lu et al., Cryst. Growth Des.,
2007, 7, 459-464). Additives that template the growth of the
nanoparticles and prevent their aggregation are commonly used in these
techniques.

[0009] The most commonly used additives are surfactants. The surfactants
form reverse-micelles or microemulsions inside which the metal
nanoparticles grow. The size of the nanoparticles is limited by the size
of the reverse-micelles (see, for example, A. Martino et al., Applied
Catalysis a-General, 1997, 161, 235-248).

[0013] There is a need for new methods for preparing metal nanoparticles.

[0014] It is an object of the present invention to go some way to meeting
this need; and/or to at least provide the public with a useful choice.

[0015] Other objects of the invention may become apparent from the
following description which is given by way of example only.

[0016] Any discussion of documents, acts, materials, devices, articles or
the like which has been included in the present specification is solely
for the purpose of providing a context for the present invention. It is
not to be taken as an admission that any or all of these matters form
part of the prior art base or were common general knowledge in the field
relevant to the present invention as it existed before the priority date.

SUMMARY OF THE INVENTION

[0017] In a first aspect, the present invention provides a metal
nanoparticle-phosphopeptide complex comprising: [0018] a metal
nanoparticle; and [0019] a phosphopeptide comprising two or more
contiguous peptide motifs and two or more phosphorus-containing groups
capable of interacting with the surface of the metal nanoparticle,
[0020] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0021]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs.

[0022] The metal nanoparticle comprises at least one metal. In one
embodiment, the metal nanoparticle comprises a single metal. In another
embodiment, the metal nanoparticle comprises a mixture of two or more
metals.

[0023] In one embodiment, the metal is selected from the metals in groups
3 to 12 of the periodic table. In another embodiment, the metal is
selected from the metals in groups 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7
to 12, or 8 to 12 of the periodic table. In another embodiment, the metal
is selected from the metals in groups 3 to 11, 4 to 11, 5 to 11, 6 to 11,
7 to 11, or 8 to 11 of the periodic table. In one exemplary embodiment,
the metal is selected from the metals in groups 8 to 11 of the periodic
table. In one embodiment, the metal is selected from the metals in
periods 4 to 6 of the periodic table. In one exemplary embodiment, the
metal is selected from the metals in periods 4 to 6 and groups 8 to 11 of
the periodic table.

[0024] In one specifically contemplated embodiment, the metal is selected
from the group consisting of iron, cobalt, nickel, copper, ruthenium,
rhodium, palladium, silver, iridium, platinum, and gold. In one
specifically contemplated embodiment, the metal is selected from the
group consisting of iron, cobalt, ruthenium, rhodium, palladium, silver,
platinum, and gold. In another specifically contemplated embodiment, the
metal is selected from the group consisting of iron, cobalt, ruthenium,
rhodium, palladium, silver, and gold. In another specifically
contemplated embodiment, the metal is selected from the group consisting
of iron, ruthenium, rhodium, palladium, silver, and gold. In another
specifically contemplated embodiment, the metal is selected from the
group consisting of iron, ruthenium, palladium, and gold. In another
specifically contemplated embodiment, the metal is iron.

[0025] In one embodiment, the metal nanoparticle is an iron nanoparticle.
In one embodiment, the iron nanoparticle is an iron oxide nanoparticle.
In one embodiment, the size of the iron oxide nanoparticle is from about
5 nm to about 8 nm.

[0026] In another embodiment, the iron nanoparticle is an iron-iron oxide
core-shell nanoparticle. In one embodiment, the size of the iron-iron
oxide core-shell nanoparticle is from about 8 nm to about 25 nm. In
another embodiment, the size of the iron-iron oxide core-shell
nanoparticle is from about 15 nm to about 25 nm.

[0027] In one embodiment, the metal nanoparticle is a cobalt nanoparticle.

[0028] In one embodiment, the metal nanoparticle is a nickel nanoparticle.

[0029] In one embodiment, the metal nanoparticle is a copper nanoparticle.

[0030] In one embodiment, the metal nanoparticle is a ruthenium
nanoparticle. In one embodiment, the size of the ruthenium nanoparticle
is from about 20 nm to 100 nm.

[0031] In one embodiment, the metal nanoparticle is a rhodium
nanoparticle.

[0032] In one embodiment, the metal nanoparticle is a palladium
nanoparticle. In one embodiment, the size of the palladium nanoparticle
is from about 3 nm to about 7 nm. In another embodiment, the size of the
palladium nanoparticle is about 5 nm.

[0033] In one embodiment, the metal nanoparticle is a silver nanoparticle.

[0034] In one embodiment, the metal nanoparticle is an iridium
nanoparticle.

[0035] In one embodiment, the metal nanoparticle is a platinum
nanoparticle.

[0036] In one embodiment, the metal nanoparticle is a gold nanoparticle.
In one embodiment, the size of the gold nanoparticle is from about 3 nm
to about 5 nm. In another embodiment, the size of the gold nanoparticle
is about 4 nm.

[0037] In one embodiment, the metal nanoparticle exhibits ferromagnetic
behaviour at room temperature. In another embodiment, the metal
nanoparticle exhibits ferrimagnetic behaviour at room temperature. In
another embodiment, the metal nanoparticle exhibits super-paramagnetic
behaviour at room temperature. In one embodiment, the metal nanoparticle
comprises iron, cobalt, nickel, or a mixture of any two or more thereof.
In another embodiment, the metal nanoparticle comprises iron or a mixture
of iron and cobalt, iron and nickel, or iron, cobalt, and nickel. In
another embodiment, the metal nanoparticle comprises iron. In another
embodiment, the metal nanoparticle is an iron nanoparticle.

[0038] In one embodiment, the molar ratio of metal to phosphopeptide is
more than about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1,
or 20:1. In one embodiment, the molar ratio is from about 1:1 to 25:1,
1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 5:1 to 25:1, 5:1 to
20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1,
15:1 to 25:1, 15:1 to 20:1, or 20:1 to 25:1.

[0039] In one embodiment, the phosphorus-containing group comprises a
phosphate or phosphonate group.

[0040] In one embodiment, the phosphopeptide favours a helical structure
in solution.

[0041] In one embodiment, the amino acid sequence of the two or more
contiguous peptide motifs is such that the contiguous peptide motifs
favour an amphipathic helical structure in solution.

[0042] In one embodiment, the amino acid sequence of the two or more
contiguous peptide motifs is such that the phosphopeptide favours an
amphipathic helical structure in solution.

[0043] In one embodiment, each peptide motif is 3 or more amino acids in
length. In one embodiment, each peptide motif is 3 to 7 amino acids in
length. In another embodiment, each peptide motif is 3, 4, or 5 amino
acids in length. In another embodiment, each peptide motif is 3 amino
acids in length.

[0044] In one embodiment, the two or more phosphorus-containing groups are
bound to amino acids at the equivalent position in each peptide motif.

[0045] In one embodiment, each amino acid is selected from one of the
following categories: polar amino acids, non polar amino acids,
hydrophobic amino acids, and non hydrophobic amino acids; and the amino
acids at the equivalent position in each peptide motif are selected from
the same category of amino acids.

[0046] In one embodiment, each amino acid is independently an amino acid
residue of the formula (II):

##STR00001##

[0047] wherein: [0048] R1 is selected from the group consisting of
hydrogen, C1-6alkyl, C2-6alkenyl, and C2-6alkynyl, each of
which is optionally substituted with one or more substituents
independently selected from the group consisting of hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkoxy,
cyano, nitro, amino, and carboxyl; [0049] R2 is selected from the
group consisting of hydrogen, C1-6alkyl, C2-6alkenyl, C2-6
alkynyl, C3-10cycloalkyl, C1-6alkylC3-10cycloalkyl,
C2-6alkenylC3-10cycloalkyl, C2-6
alkynylC3-10cycloalkyl, C3-10cycloalkenyl,
C1-6alkylC3-10cycloalkenyl,
C2-6alkenylC3-10cycloalkenyl,
C2-6alkynylC3-10cycloalkenyl, aryl, C1-6alkylaryl,
C2-6alkenylaryl, C2-6alkynylaryl, heteroaryl,
C1-6alkylheteroaryl, C2-6alkenylheteroaryl,
C2-6alkynylheteroaryl, heterocyclyl, C1-6alkylheterocyclyl,
C2-6alkenylheterocyclyl, and C2-6alkynylheterocyclyl, each of
which is optionally substituted with one or more substituents
independently selected from the group consisting of C1-6alkyl,
C2-6alkenyl, C2-6alkynyl, hydroxyl, C1-6alkyoxy, thiol,
C1-6alkylthio, halo, C1-6haloalkyl, C1-6haloalkoxy, acyl,
amino, amido, acylamino, carboxyl, acyloxy, guanidino, urea, carbonate,
thiourea, cyano, nitro, nitroso, azide, cyanate, thiocyanate, and
isocyanate; [0050] R3 is selected from the group consisting of
hydrogen, C1-6alkyl, C2-6alkenyl, and C2-6alkynyl, each of
which is optionally substituted with one or more substituents
independently selected from the group consisting of hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkoxy,
cyano, nitro, amino, and carboxyl; [0051] or R1 and R2 together
with nitrogen atom and carbon atom to which they are attached form a 5-
or 6-membered heterocyclyl ring optionally substituted with one or more
substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, cyano, and nitro; [0052] or R2 and R3
together with the carbon atom to which they are attached form a 5- or
6-membered cycloalkyl, cycloalkenyl, or heterocyclyl ring optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy, acyl, amino, amido, acylamino,
carboxyl, acyloxy, guanidino, urea, carbonate, thiourea, cyano, nitro,
nitroso, azide, cyanate, thiocyanate, and isocyanate; [0053] m is an
integer from 0 to 2 and p is 0, or m is 0 and p is an integer from 0 to
2.

[0054] In one embodiment, each phosphorus-containing group is bound to an
amino acid residue of the formula (II).

[0055] In one embodiment, each phosphorus-containing group is bound to
R2, the optionally substituted ring formed when R1 and R2
are taken together with nitrogen atom and carbon atom to which they are
attached, or the optionally substituted ring formed when R2 and
R3 are taken together with the carbon atom to which they are
attached.

[0056] In one embodiment, the phosphorus-containing group is selected from
the group consisting of phosphate, C1-6alkylphosphate,
C2-6alkenylphosphate, C2-6alkynylphosphate, arylphosphate,
C1-6alkylarylphosphate, C2-6alkenylarylphosphate,
C2-6alkynylarylphosphate, phosphonate, C1-6alkylphosphonate,
C2-6alkenylphosphonate, C2-6alkynylphosphonate,
arylphosphonate, C1-6alkylarylphosphonate,
C2-6alkenylarylphosphonate, C2-6alkynylarylphosphonate. In one
embodiment, the phosphorus-containing group is selected from the group
consisting of phosphate, phosphonate, C1-6alkylphosphate, and
C1-6alkylphosphonate.

[0057] In one embodiment, each amino acid is independently an amino acid
residue of the formula (II) wherein: [0058] R1 is selected from
the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl, and
C2-6alkynyl, each of which is optionally substituted with one or
more halo; [0059] R2 is selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C1-6alkylaryl,
and C1-6alkylheteroaryl, wherein each C1-6alkyl,
C2-6alkenyl, and C2-6alkynyl is substituted with hydroxyl,
thiol, amino, amido, carboxyl, or guanidino, and optionally substituted
with one or more substituents independently selected from the group
consisting of hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio,
halo, C1-6haloalkyl, C1-6haloalkoxy, cyano, and nitro, each
C1-6alkylaryl is substituted with hydroxyl, thiol, or amino, and
optionally substituted with one or more substituents independently
selected from the group consisting of C1-6alkyl, C2-6alkenyl,
C2-6alkynyl, hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio,
halo, C1-6haloalkyl, C1-6haloalkoxy, amino, cyano, and nitro,
and each C1-6alkylheteroaryl is optionally substituted with one or
more substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, amino, cyano, and nitro; [0060] R3 is selected
from the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl,
and C2-6alkynyl, each of which is optionally substituted with one or
more halo; [0061] or R1 and R2 together with nitrogen atom and
carbon atom to which they are attached form a 5- or 6-membered
heterocyclyl ring substituted with hydroxyl or thiol and optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy, cyano, and nitro; [0062] or
R2 and R3 together with the carbon atom to which they are
attached form a 5- or 6-membered cycloalkyl or cycloalkenyl ring
substituted with hydroxyl or thiol and optionally substituted with one or
more substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, or a 5- or 6-membered heterocyclyl ring optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy; and [0063] m is 0 or 1 and p is
0, or m is 0 and p is 0 or 1.

[0064] In one embodiment, each phosphorus-containing group is bound to
R2, the optionally substituted ring formed when R1 and R2
are taken together with nitrogen atom and carbon atom to which they are
attached, or the optionally substituted ring formed when R2 and
R3 are taken together with the carbon atom to which they are
attached.

[0065] In one embodiment, the phosphorus-containing group is selected from
the group consisting of phosphate, C1-6alkylphosphate,
C2-6alkenylphosphate, C2-6alkynylphosphate, arylphosphate,
C1-6alkylarylphosphate, C2-6alkenylarylphosphate,
C2-6alkynylarylphosphate, phosphonate, C1-6alkylphosphonate,
C2-6alkenylphosphonate, C2-6alkynylphosphonate,
arylphosphonate, C1-6alkylarylphosphonate,
C2-6alkenylarylphosphonate, C2-6alkynylarylphosphonate. In one
embodiment, the phosphorus-containing group is selected from the group
consisting of phosphate, phosphonate, C1-6alkylphosphate, and
C1-6alkylphosphonate.

[0066] In another embodiment, each amino acid is independently an amino
acid residue of the formula (II) wherein: [0067] R1 and R3
are each hydrogen; [0068] R2 is selected from the group consisting
of C1-6alkyl, C1-6alkylaryl, and C1-6alkylheteroaryl,
wherein each C1-6alkyl is substituted with hydroxyl, thiol, amino,
amido, carboxyl, or guanidino, and each C1-6alkylaryl is substituted
with hydroxyl; and [0069] m is 0 and p is 0.

[0070] In one embodiment, each phosphorus-containing group is bound to
R2.

[0071] In one embodiment, the phosphorus-containing group is selected from
the group consisting of phosphate, C1-6alkylphosphate,
C2-6alkenylphosphate, C2-6alkynylphosphate, arylphosphate,
C1-6alkylarylphosphate, C2-6alkenylarylphosphate,
C2-6alkynylarylphosphate, phosphonate, C1-6alkylphosphonate,
C2-6alkenylphosphonate, C2-6alkynylphosphonate,
arylphosphonate, C1-6alkylarylphosphonate,
C2-6alkenylarylphosphonate, C2-6alkynylarylphosphonate. In one
embodiment, the phosphorus-containing group is selected from the group
consisting of phosphate, phosphonate, C1-6alkylphosphate, and
C1-6alkylphosphonate.

[0072] In one embodiment, each amino acid is independently a natural amino
acid; or an unnatural amino acid residue of the formula (II) wherein:
[0073] R1 and R3 are each hydrogen; [0074] R2 is
C1-6alkylheteroaryl; and [0075] m is 0 and p is 0.

[0076] In one embodiment, each phosphorus-containing group is bound to the
oxygen atom of a hydroxyl group in a serine, threonine, or tyrosine
residue; the nitrogen atom of an imidazole ring in a histidine residue;
or the heteroaryl group of an amino acid residue of the formula (II)
wherein R2 is C1-6alkylheteroaryl.

[0077] In one embodiment, each phosphorus-containing group is bound to the
oxygen atom of a hydroxyl group in a serine, threonine, or tyrosine
residue; or the heteroaryl group of an amino acid residue of the formula
(II) wherein R2 is C1-6alkylheteroaryl.

[0078] In one embodiment, the heteroaryl group is a triazole ring. In one
embodiment, the triazole ring is a 1,2,3-triazole ring. In one
embodiment, the 1,2,3-triazole ring is 1,4-substituted.

[0079] In one embodiment, each phosphorus-containing group bound to the
oxygen atom of a hydroxyl group in a serine, threonine, or tyrosine
residue is a --P(O)(OH)2 group.

[0080] In one embodiment, each phosphorus-containing group bound to the
heteroaryl group of an amino acid residue of the formula (II) wherein
R2 is C1-6alkylheteroaryl is a C1-6alkylphosphonate. In
one embodiment, the heteroaryl group is a triazole ring. In one
embodiment, the triazole ring is a 1,2,3-triazole ring.

[0081] In one embodiment, the phosphopeptide comprises:

[0082] an amino acid sequence of the formula (I):

Xaa1-Xaa2-Xaa3n (I) [0083] wherein: [0084] n is
an integer from 2 to 50; [0085] Xaa1, Xaa2, and Xaa3 at
each instance of n are each independently an amino acid residue of the
formula (II):

[0085] ##STR00002## [0086] wherein: [0087] R1 is selected from
the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl, and
C2-6alkynyl, each of which is optionally substituted with one or
more substituents independently selected from the group consisting of
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkoxy, cyano, nitro, amino, and carboxyl; [0088] R2 is
selected from the group consisting of hydrogen, C1-6alkyl,
C2-6alkenyl, C2-6alkynyl, C3-10cycloalkyl,
C1-6alkylC3-10cycloalkyl, C2-6alkenylC3-10cycloalkyl,
C2-6alkynylC3-10cycloalkyl, C3-10cycloalkenyl,
C1-6alkylC3-10cycloalkenyl,
C2-6alkenylC3-10cycloalkenyl,
C2-6alkynylC3-10cycloalkenyl, aryl, C1-6alkylaryl,
C2-6alkenylaryl, C2-6alkynylaryl, heteroaryl,
C1-6alkylheteroaryl, C2-6alkenylheteroaryl,
C2-6alkynylheteroaryl, heterocyclyl, C1-6alkylheterocyclyl,
C2-6alkenylheterocyclyl, and C2-6alkynylheterocyclyl, each of
which is optionally substituted with one or more substituents
independently selected from the group consisting of C1-6alkyl,
C2-6alkenyl, C2-6alkynyl, hydroxyl, C1-6alkyoxy, thiol,
C1-6alkylthio, halo, C1-6haloalkyl, C1-6haloalkoxy, acyl,
amino, amido, acylamino, carboxyl, acyloxy, guanidino, urea, carbonate,
thiourea, cyano, nitro, nitroso, azide, cyanate, thiocyanate, and
isocyanate; [0089] R3 is selected from the group consisting of
hydrogen, C1-6alkyl, C2-6alkenyl, and C2-6alkynyl, each of
which is optionally substituted with one or more substituents
independently selected from the group consisting of hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkoxy,
cyano, nitro, amino, and carboxyl; [0090] or R1 and R2 together
with nitrogen atom and carbon atom to which they are attached form a 5-
or 6-membered heterocyclyl ring optionally substituted with one or more
substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, cyano, and nitro; [0091] or R2 and R3
together with the carbon atom to which they are attached form a 5- or
6-membered cycloalkyl, cycloalkenyl, or heterocyclyl ring optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy, acyl, amino, amido, acylamino,
carboxyl, acyloxy, guanidino, urea, carbonate, thiourea, cyano, nitro,
nitroso, azide, cyanate, thiocyanate, and isocyanate; [0092] m is an
integer from 0 to 2 and p is 0, or m is 0 and p is an integer from 0 to
2; and [0093] provided that Xaa1, Xaa2, and Xaa3
respectively, at each instance of n, have similar structural and/or
electronic properties; two or more phosphorus-containing groups capable
of interacting with the surface of the metal nanoparticle, [0094] wherein
each phosphorus-containing group is bound to R2, the optionally
substituted ring formed when R1 and R2 are taken together with
nitrogen atom and carbon atom to which they are attached, or the
optionally substituted ring formed when R2 and R3 are taken
together with the carbon atom to which they are attached of an amino acid
in the amino acid sequence of formula (I); and optionally a group that
mitigates aggregation of the metal nanoparticle-phosphopeptide complex
with metal nanoparticles or other metal nanoparticle-phosphopeptide
complexes; [0095] wherein each group that mitigates aggregation is bound
to R2, the optionally substituted ring formed when R1 and
R2 are taken together with nitrogen atom and carbon atom to which
they are attached, or the optionally substituted ring formed when R2
and R3 are taken together with the carbon atom to which they are
attached of an amino acid in the amino acid sequence of formula (I).

[0096] In one embodiment, and Xaa1, Xaa2, or Xaa3 that is
bound to a phosphorus-containing group is not adjacent to another
Xaa1, Xaa2, or Xaa3 that is bound to a
phosphorus-containing group.

[0097] In one embodiment, Xaa1, Xaa2, and Xaa3,
respectively, at each instance of n have similar structural and
electronic properties.

[0098] In one embodiment, the phosphorus-containing group is selected from
the group consisting of phosphate, phosphonate, C1-6alkylphosphate,
and C1-6alkylphosphonate. In one embodiment, the
phosphorus-containing group is phosphate or phosphonate.

[0099] In one embodiment, the group that mitigates aggregation is selected
from the group consisting of sulfate, C1-6alkylsulfate, sulfonate,
C1-6alkylsulfonate, poly(ethylene oxide), poly(betaine),
poly(saccharide), and a charged peptide. In another embodiment, the group
that mitigates aggregation is selected from the group consisting of
sulfate, C1-6alkylsulfate, sulfonate, and C1-6alkylsulfonate.

[0100] In one embodiment, one or more of the amino acids of each peptide
motif are natural amino acids. In another embodiment, two or more of the
amino acids of each peptide motif are natural amino acids. In one
embodiment, the amino acid is N- or O-bound to a phospho group.

[0101] In another embodiment, one or more of the amino acids of each
peptide motif are natural amino acids. In another embodiment, two or more
of the amino acids of each peptide motif are natural amino acids. In
another embodiment, all of the amino acids of each peptide motif are
natural amino acids; or all of the amino acids of each peptide motif are
natural amino acids, except any amino acid bound to a phosphorus
containing group. In another embodiment, all of the amino acids of each
peptide motif are natural amino acids.

[0102] In a further aspect, the present invention provides a
phosphopeptide as defined in any of the embodiments described herein.

[0103] In a further aspect, the present invention provides a composition
comprising a plurality of metal nanoparticles and a phosphopeptide of the
present invention. In one embodiment, the metal is as defined in any of
the preceding embodiments.

[0104] In a further aspect, the present invention provides a composition
comprising a plurality of metal nanoparticle-phosphopeptide complexes of
the present invention. In one embodiment, the metal is as defined in any
of the preceding embodiments.

[0105] In one embodiment, the composition further comprises a solvent in
which the metal nanoparticle-phosphopeptide complexes are suspended. In
one embodiment, the suspension is stable for at least one day. In one
embodiment, the solvent is water or an alcohol. In one embodiment, the
alcohol is ethanol. In one embodiment, the solvent is water.

[0106] In another embodiment, the composition further comprises a
pharmaceutically acceptable carrier, excipient, or diluent.

[0107] In a further aspect, the present invention provides a method for
preparing metal nanoparticles, the method comprising contacting [0108]
a metal compound; and [0109] a phosphopeptide comprising two or more
contiguous peptide motifs and two or more phosphorus-containing groups
capable of interacting with the surface of the metal nanoparticle,
[0110] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0111]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs; in a liquid reaction medium under
conditions that form metal nanoparticles.

[0112] In a further aspect, the present invention provides a method for
preparing a metal nanoparticle-phosphopeptide complex, the method
comprising contacting [0113] a metal compound; and [0114] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the metal nanoparticle, [0115] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0116] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs; in a liquid reaction medium under conditions that form a metal
nanoparticle-phosphopeptide complex.

[0117] In one embodiment, the metal is as defined in any of the preceding
embodiments.

[0118] In one embodiment, the metal compound comprises a metal cation.

[0119] In one embodiment, the method comprises contacting two or more
metal compounds. In one embodiment, the at least two of the two or more
metal compounds comprise different metals.

[0120] In one embodiment, the method comprises reducing the metal compound
with a reducing agent in the presence of the phosphopeptide complex to
form the metal nanoparticle-phosphopeptide complex.

[0121] In another embodiment, the method comprises precipitating metal
nanoparticles from the metal compound in the presence of the
phosphopeptide to form the metal nanoparticle-phosphopeptide complex.

[0122] In a further aspect, the present invention provides a method for
preparing metal nanoparticles, the method comprising contacting [0123]
a metal compound; [0124] a phosphopeptide comprising two or more
contiguous peptide motifs and two or more phosphorus-containing groups
capable of interacting with the surface of the metal nanoparticle,
[0125] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0126]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs; and [0127] a reducing agent in a
liquid reaction medium to form metal nanoparticles.

[0128] In one embodiment, the metal is as defined in any of the preceding
embodiments.

[0129] In a further aspect, the present invention provides a method for
preparing a metal nanoparticle-phosphopeptide complex, the method
comprising contacting [0130] a metal compound; [0131] a phosphopeptide
comprising two or more contiguous peptide motifs and two or more
phosphorus-containing groups capable of interacting with the surface of
the metal nanoparticle, [0132] wherein the amino acids at the equivalent
position in each peptide motif have similar structural and/or electronic
properties, and [0133] wherein each phosphorus-containing group is bound
to an amino acid in the two or more contiguous peptide motifs; and
[0134] a reducing agent in a liquid reaction medium to form a metal
nanoparticle-phosphopeptide complex.

[0135] In one embodiment, the metal is as defined in any of the preceding
embodiments.

[0138] The phosphopeptide is as defined in any of the embodiments
described herein. In one embodiment, the molar concentration of
phosphopeptide relative to metal is low.

[0139] In one embodiment, the molar ratio of metal to phosphopeptide is
more than about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1,
or 20:1. In one embodiment, the molar ratio is from about 1:1 to 25:1,
1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 5:1 to 25:1, 5:1 to
20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1,
15:1 to 25:1, 15:1 to 20:1, or 20:1 to 25:1.

[0140] In one embodiment, the reducing agent is a metal hydride. In one
embodiment, the metal hydride is a metal borohydride. In one embodiment,
the metal borohydride is sodium borohydride.

[0141] In one embodiment, the methods comprise mixing the metal compound,
phosphopeptide, and reducing agent in the liquid reaction medium.

[0142] In one embodiment, the methods further comprise recovering the
product metal nanoparticles or metal nanoparticle-phosphopeptide complex.

[0143] In one embodiment, the nanoparticle is an iron nanoparticle. In one
embodiment, the iron nanoparticle is an iron-iron oxide core-shell
nanoparticle. In one embodiment, the size of the iron-iron oxide
core-shell nanoparticle is from about 8 nm to about 25 nm. In another
embodiment, the size of the iron-iron oxide core-shell nanoparticle is
from about 15 nm to about 25 nm.

[0144] In another embodiment, the iron nanoparticle is an iron oxide
nanoparticle. In one embodiment, the size of the iron oxide nanoparticle
is about 8 nm.

[0145] In one embodiment, the metal nanoparticle is a cobalt nanoparticle.

[0146] In one embodiment, the metal nanoparticle is a nickel nanoparticle.

[0147] In one embodiment, the metal nanoparticle is a copper nanoparticle.

[0148] In one embodiment, the metal nanoparticle is a ruthenium
nanoparticle. In one embodiment, the size of the ruthenium nanoparticle
is from about 20 nm to 100 nm.

[0149] In one embodiment, the metal nanoparticle is a rhodium
nanoparticle.

[0150] In one embodiment, the metal nanoparticle is a palladium
nanoparticle. In one embodiment, the size of the palladium nanoparticle
is from about 3 nm to about 7 nm. In another embodiment, the size of the
palladium nanoparticle is about 5 nm.

[0151] In one embodiment, the metal nanoparticle is a silver nanoparticle.

[0152] In one embodiment, the metal nanoparticle is an iridium
nanoparticle.

[0153] In one embodiment, the metal nanoparticle is a platinum
nanoparticle.

[0154] In one embodiment, the metal nanoparticle is a gold nanoparticle.
In one embodiment, the size of the gold nanoparticle is from about 3 nm
to about 5 nm. In another embodiment, the size of the gold nanoparticle
is about 4 nm.

[0155] In one embodiment, the metal nanoparticles are substantially
monodisperse.

[0156] In one embodiment, the liquid reaction medium is water.

[0157] In one embodiment, the methods are carried out at ambient
temperature.

[0158] In one embodiment, the reaction is carried out for a period of time
from 2 minutes to 12 hours, 2 minutes to 3 hours, 2 minutes to 1 hour, 5
minutes to 12 hours, 5 minutes to 3 hours, 5 minutes to 1 hour, 10
minutes to 12 hours, 10 minutes to 3 hours, 10 minutes to 1 hour.

[0159] In a further aspect, the present invention provides a method for
preparing metal nanoparticles, the method comprising contacting [0160]
a metal compound; and [0161] a phosphopeptide comprising two or more
contiguous peptide motifs and two or more phosphorus-containing groups
capable of interacting with the surface of the metal nanoparticle,
[0162] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0163]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs; and in a liquid reaction medium
under conditions that precipitate metal nanoparticles.

[0164] In a further aspect, the present invention provides a method for
preparing a metal nanoparticle-phosphopeptide complex, the method
comprising contacting [0165] a metal compound; and [0166] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the metal nanoparticle, [0167] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0168] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs; and in a liquid reaction medium under conditions that precipitate
a metal nanoparticle-phosphopeptide complex.

[0169] In one embodiment, the metal is as defined in any of the preceding
embodiments.

[0170] In one embodiment, the method comprises contacting two or more
metal compounds. In one embodiment, at least two of the two or more metal
compounds comprise different metals.

[0171] In one embodiment, the method comprises co-precipitating two or
more metal compounds in the presence of the phosphopeptide to form the
metal nanoparticle phosphopeptide complex.

[0172] In one embodiment, at least one of the metal compounds comprises
iron.

[0173] In one embodiment, the metal compound is a metal salt. In one
embodiment, the metal salt is as defined in any of the preceding
embodiments.

[0174] The phosphopeptide is as defined in any of the embodiments
described herein. In one embodiment, the molar concentration of
phosphopeptide relative to metal is low.

[0175] In one embodiment, the molar ratio of metal to phosphopeptide is
more than about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1,
or 20:1. In one embodiment, the molar ratio is from about 1:1 to 25:1,
1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 5:1 to 25:1, 5:1 to
20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1,
15:1 to 25:1, 15:1 to 20:1, or 20:1 to 25:1.

[0176] In one embodiment, the methods comprise mixing the metal compound
and phosphopeptide in the liquid reaction medium.

[0177] In one embodiment, the methods further comprise recovering the
product metal nanoparticles or metal nanoparticle-phosphopeptide complex.

[0178] In one embodiment, the metal nanoparticle is a metal oxide, metal
hydroxide, or metal chalcogenide nanoparticle.

[0179] In one embodiment, the method comprises contacting one or more
metal compounds, a phosphopeptide, and hydroxide or chalcogen anions in
the liquid reaction medium. In one embodiment, the chalcogen is sulfur.

[0180] In one embodiment, the liquid reaction medium comprises water. In
one embodiment, the liquid reaction medium is water.

[0181] In one embodiment, the liquid reaction medium comprises base.

[0182] In one embodiment, the metal nanoparticles are substantially
monodisperse.

[0183] In one embodiment, the methods are carried out at ambient
temperature.

[0184] In a further aspect, the present invention provides a method for
preparing iron nanoparticles, the method comprising contacting [0185]
iron (II); [0186] iron (III); [0187] a phosphopeptide comprising two or
more contiguous peptide motifs and two or more phosphorus-containing
groups capable of interacting with the surface of the iron nanoparticle,
[0188] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0189]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs; and [0190] a base in a liquid
reaction medium to form iron nanoparticles.

[0191] In a further aspect, the present invention provides a method for
preparing an iron nanoparticle-phosphopeptide complex, the method
comprising contacting [0192] iron (II); [0193] iron (III); [0194] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the iron nanoparticle, [0195] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0196] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs; and [0197] a base in a liquid reaction medium to provide an
iron nanoparticle-phosphopeptide complex.

[0198] In one embodiment, iron (II) is provided to the liquid reaction
mixture in the form of an iron (II) compound. In one embodiment, iron
(III) is provided to the liquid reaction mixture in the form of an iron
(III) compound. In one embodiment iron (II) and iron (III) are provided
to the liquid reaction mixture in the form of an iron (II) compound and
an iron (III) compound, respectively.

[0199] In one embodiment, the iron (II) compound is an iron (II) salt. In
one embodiment, the iron (III) compound is an iron (III) salt. In one
embodiment, the iron (II) compound is an iron (II) salt and the iron
(III) compound is an iron (III) salt.

[0200] In one embodiment, the iron (II) compound is iron (II) sulfate and
the iron (III) compound is iron (III) chloride.

[0201] In one embodiment, the base is ammonia.

[0202] The phosphopeptide is as defined in any of the embodiments
described herein. In one embodiment, the molar ratio of
phosphorus-containing groups to iron is less than 1:1. In another
embodiment, the molar ratio of phosphorus-containing groups to iron (III)
is less than 1:1.

[0203] In one embodiment, the methods comprise mixing the iron (II), iron
(III), phosphopeptide, and base in the liquid reaction medium.

[0204] In one embodiment, the methods further comprise recovering the
product iron nanoparticles or iron nanoparticle-phosphopeptide complex.

[0205] In one embodiment, the iron nanoparticle is an iron oxide
nanoparticle. In one embodiment, the iron oxide nanoparticle is about 5
nm.

[0206] In one embodiment, the iron nanoparticles are substantially
monodisperse.

[0207] In one embodiment, the liquid reaction medium comprises water. In
one embodiment, the liquid reaction medium is water.

[0208] In one embodiment, the methods are carried out at ambient
temperature.

[0209] In one embodiment, the reaction is carried out for a period of time
from 2 minutes to 12 hours, 2 minutes to 3 hours, 2 minutes to 1 hour, 5
minutes to 12 hours, 5 minutes to 3 hours, 5 minutes to 1 hour, 10
minutes to 12 hours, 10 minutes to 3 hours, 10 minutes to 1 hour.

[0210] In a further aspect, the present invention provides metal
nanoparticles prepared by a method of the present invention.

[0211] In a further aspect, the present invention provides a metal
nanoparticle-phosphopeptide complex prepared by a method of the present
invention.

[0212] In a further aspect, the present invention provides a use of a
metal nanoparticle-phosphopeptide complex of the present invention in the
manufacture of a medicament for treating cancer. In a further aspect, the
present invention provides a use of a metal nanoparticle-phosphopeptide
complex of the present invention in the manufacture of a contrast agent
for contrast enhancement in medical imaging.

[0213] In a further aspect, the present invention provides a metal
nanoparticle-phosphopeptide complex of the present invention for use in
treating cancer. In a further aspect, the present invention provides a
metal nanoparticle-phosphopeptide complex of the present invention for
use in contrast enhancement in medical imaging.

[0214] In a further aspect, the present invention provides a method of
treating cancer comprising administering an effective amount of a metal
nanoparticle-phosphopeptide complex of the present invention to a patient
in need thereof, and applying an alternating magnetic field to heat the
nanoparticles. In a further aspect, the present invention provides a
method of imaging comprising administering an effective amount of a metal
nanoparticle-phosphopeptide complex of the present invention to a patient
in need thereof, and imaging the patient.

[0215] In a further aspect, the present invention provides a use of the
metal nanoparticles of the present invention in the manufacture of a
medicament for treating cancer. In a further aspect, the present
invention provides a use of the metal nanoparticles of the present
invention in the manufacture of a contrast agent for contrast enhancement
in medical imaging.

[0216] In a further aspect, the present invention provides metal
nanoparticles of the present invention for use in treating cancer. In a
further aspect, the present invention provides metal nanoparticles for
use in contrast enhancement in medical imaging.

[0217] In a further aspect, the present invention provides a method of
treating cancer comprising administering an effective amount of metal
nanoparticles of the present invention to a patient in need thereof, and
applying an alternating magnetic field to heat the nanoparticles. In a
further aspect, the present invention provides a method of imaging
comprising administering an effective amount of metal nanoparticles of
the present invention to a patient in need thereof, and imaging the
patient.

[0218] In a further aspect, the present invention provides a kit for
preparing a therapeutic or diagnostic agent comprising: [0219] a metal
compound; and [0220] a phosphopeptide comprising two or more contiguous
peptide motifs and two or more phosphorus-containing groups capable of
interacting with the surface of the metal nanoparticle, [0221] wherein
the amino acids at the equivalent position in each peptide motif have
similar structural and/or electronic properties, and [0222] wherein each
phosphorus-containing group is bound to an amino acid in the two or more
contiguous peptide motifs.

[0223] In one embodiment, the kit further comprises instructions for
preparing a metal nanoparticle-phosphopeptide complex by a method of the
present invention.

[0224] In another embodiment, the kit further comprises a reducing agent.
In one embodiment, the reducing agent is sodium borohydride.

[0225] In one embodiment, the kit comprises a liquid medium in which the
metal nanoparticle-phosphopeptide complex is prepared.

[0226] In a further aspect, the present invention provides a kit for
preparing a therapeutic or diagnostic agent comprising: [0227] a metal
nanoparticle-phosphopeptide complex of the present invention.

[0228] In one embodiment, the kit comprises a liquid medium in which the
metal nanoparticle-phosphopeptide complex is suspended.

[0229] In one embodiment, the therapeutic agent is for use in treating
cancer. In one embodiment, the diagnostic agent is for use as a contrast
agent in medical imaging.

[0230] In another embodiment, the kits further comprise a compound that
minimises non-specific interactions and/or inflammatory reactions in
vivo. In one embodiment, the kit further comprises instructions for
coupling the compound to the metal nanoparticle-phosphopeptide complex.

[0231] In one embodiment, the kits comprise a targeting group that has a
specific interaction with a target in vivo. In one embodiment, the
targeting group comprises an antibody that has a specific interaction
with a target antigen in vivo. In one embodiment, the target antigen is a
cell-surface receptor.

[0232] In one embodiment, the kits further comprise an activating agent to
facilitate coupling of the compound and/or targeting group to the metal
nanoparticle-phosphopeptide complex. In one embodiment, the activating
agent is an activating agent for peptide coupling. In one embodiment, the
kit further comprises instructions for coupling the compound and/or
targeting group to the metal nanoparticle-phosphopeptide complex. In one
embodiment, the kits comprise a liquid medium in which the coupling
reaction(s) are carried out.

[0233] In one embodiment, the metal nanoparticle comprises iron, cobalt,
nickel, or a mixture of any two or more thereof. In another embodiment,
the metal nanoparticle comprises iron or a mixture of iron and cobalt,
iron and nickel, or iron, cobalt, and nickel. In another embodiment, the
metal nanoparticle comprises iron. In another embodiment, the metal
nanoparticle is an iron nanoparticle.

[0234] In a further aspect, the present invention provides a metal
nanoparticle-phosphopeptide complex of the present invention for use as a
catalyst.

[0235] In a further aspect, the present invention provides a catalyst
comprising a metal nanoparticle-phosphopeptide complex of the present
invention.

[0236] In a further aspect, the present invention provides use of a metal
nanoparticle-phosphopeptide complex of the present invention as a
catalyst.

[0237] As used herein the term "and/or" means "and", or "or", or both.

[0238] The term "comprising" as used in this specification means
"consisting at least in part of". When interpreting each statement in
this specification that includes the term "comprising", features other
than that or those prefaced by the term may also be present. Related
terms such as "comprise" and "comprises" are to be interpreted in the
same manner.

[0239] As used herein the "nanoparticle" refers to any particle less than
1000 nanometres in size. In one embodiment, the nanoparticle is less than
1000 nm, 750 nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm,
175 nm, 150 nm, 125 nm, or 100 nm. In one embodiment, the nanoparticle is
less than 100 nm. Those persons skilled in the art will appreciate that
references to nanoparticles in this specification it also include
nanocrystals, even though a nanocrystal may have a higher degree of
crystallinity than a nanoparticle.

[0240] The term "metal nanoparticle" as used herein refers to a
nanoparticle that comprises metal. In one embodiment, the metal
nanoparticle comprises metal, metal oxide, metal hydroxide, metal
chalcogenide, or a mixture of metal and metal oxide, metal and metal
hydroxide, metal and metal chalcogenide, or metal, metal oxide, and metal
hydroxide. In another embodiment, the metal nanoparticle is substantially
composed of metal, metal oxide, metal hydroxide, metal chalcogenide, or a
mixture of metal and metal oxide, metal and metal hydroxide, metal and
metal chalcogenide, or metal, metal oxide, and metal hydroxide. In one
embodiment, the chalcogen is sulfur, selenium, or tellurium. In one
embodiment, the chalcogen is sulfur. In one embodiment, the metal
nanoparticle is substantially composed of metal, metal oxide, metal
hydroxide, or a mixture of metal and metal oxide, metal and metal
hydroxide, or metal, metal oxide, and metal hydroxide. In another
embodiment, the metal nanoparticle is substantially composed of metal,
metal oxide, or a mixture of metal and metal oxide.

[0241] A metal nanoparticle may have a "core" comprising metal surrounded
by a "shell" comprising metal oxide. The term "core" refers to the
central region of the nanoparticle. A core can substantially include a
single homogeneous material. A core may be crystalline or amorphous.
Whilst a core may be referred to as crystalline, it is understood that
the surface of the core may be amorphous or polycrystalline and that this
non-crystalline surface layer may extend a finite depth into the core.

[0242] The term "metal nanoparticle-phosphopeptide complex" as used herein
refers to a metal nanoparticle having one or more phosphopeptides on its
surface. The phosphopeptide may be adsorbed on the surface of the metal
nanoparticle. The phosphopeptide may also be partially incorporated into
the surface of the metal nanoparticle.

[0243] As used herein the "size" of a nanoparticle refers to the diameter
of the nanoparticle.

[0244] The term "peptide" as used herein alone or in combination with
other terms means a chain of two or more natural or unnatural amino acids
joined by a peptide bond.

[0245] The term "phosphopeptide" as used herein alone or in combination
with other terms means a peptide that comprises one or more
phosphorus-containing groups. In one embodiment, the phosphorus
containing group is capable of interacting with the surface of a metal
nanoparticle. In one embodiment, the phosphopeptide comprises from 4 to
500 amino acids. In another embodiment, the phosphopeptide comprises from
4 to 300 amino acids. In one embodiment, the phosphopeptide comprises
from 6 to 300 amino acids. In another embodiment, the phosphopeptide
comprises from 6 to 150 amino acids. In another embodiment, the
phosphopeptide comprises from 6 to 100 amino acids. In another
embodiment, the phosphopeptide comprises from 6 to 75 amino acids. In
another embodiment, the phosphopeptide comprises from 6 to 50 amino
acids. In another embodiment, the phosphopeptide comprises from 6 to 25
amino acids. In another embodiment, the phosphopeptide comprises from 6
to 75, from 6 to 70, from 6 to 65, from 6 to 60, from 6 to 55, from 6 to
50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25,
from 6 to 20, or from 6 to 18 amino acids.

[0246] The term "phosphate" employed alone or in combination with other
terms means, unless otherwise stated, a --OP(O)(OR1)(OR2)
group, wherein R1 and R2 are each independently selected from
the group consisting of hydrogen and a metal cation.

[0247] The term "phosphonate" employed alone or in combination with other
terms means, unless otherwise stated, a --P(O)(OR1)(OR2) group,
wherein Wand R2 are each independently selected from the group
consisting of hydrogen and a metal cation.

[0248] The term "pyrophosphate" employed alone or in combination with
other terms means, unless otherwise stated, a
--OP(O)(OR1)OP(O)(OR2)(OR3) group, wherein R1,
R2, and R3 are each independently selected from the group
consisting of hydrogen and a metal cation.

[0249] The term "sulfate" employed alone or in combination with other
terms means, unless otherwise stated, a --OS(O)2OR group, wherein R
is selected from the group consisting of hydrogen and a metal cation.

[0250] The term "sulfonate" employed alone or in combination with other
terms means, unless otherwise stated, a --S(O)2OR group, wherein R
is selected from the group consisting of hydrogen and a metal cation.

[0252] The term "alkenyl" employed alone or in combination with other
terms means, unless otherwise stated, a monovalent straight chain or
branched chain hydrocarbon group including one or more carbon-carbon
double bonds. In one embodiment, alkenyl groups comprise 2 to 6 carbon
atoms. Examples of alkenyl groups include vinyl, prop-2-enyl, crotyl,
isopent-2-enyl, 2-butadienyl, penta-2,4-dienyl, penta-1,4-dienyl, and the
like.

[0253] The term "alkynyl" employed alone or in combination with other
terms means, unless otherwise stated, a monovalent straight chain or
branched chain hydrocarbon group including one or more carbon-carbon
triple bonds. In one embodiment, alkynyl groups comprise 2 to 6 carbon
atoms. Examples of alkynyl groups include ethynyl, prop-3-ynyl,
but-3-ynyl, and the like.

[0254] The term "cycloalkyl", employed alone or in combination with other
terms means, unless otherwise stated, a monovalent saturated cyclic
hydrocarbon group. In one embodiment, cycloalkyl groups contain from 3 to
10 ring carbon atoms. In another embodiment, cycloalkyl groups comprise
from 3 to 8 ring carbon atoms. Examples of cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, and the like.

[0255] The term "cycloalkenyl", employed alone or in combination with
other terms means, unless otherwise stated, a non-aromatic monovalent
cyclic hydrocarbon group containing one or more carbon-carbon double
bonds. In one embodiment, cycloalkenyl groups contain from 3 to 10 ring
carbon atoms. In another embodiment, cycloalkenyl groups comprise 3 to 8
ring carbon atoms. Examples of cycloalkenyl groups include, but are not
limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl,
cycloheptentyl, cyclooctenyl, and the like.

[0259] The term "aliphatic" employed alone or in combination with other
terms means, unless otherwise stated, straight chain or branched chain
saturated or unsaturated hydrocarbon group. Those skilled in the art will
appreciate that aliphatic groups include alkyl, alkenyl, and alkynyl
groups.

[0260] The term "heteroaliphatic" employed alone or in combination with
other terms means, unless otherwise stated, means an aliphatic group
wherein one or more of the carbon atoms in the main hydrocarbon chain are
replaced with heteroatoms independently selected from the group
consisting of oxygen, nitrogen, and sulfur. Examples of heteroaliphatic
groups include alkoxyalkyl and alkylthioalkyl groups, and the like.

[0261] The term "alicyclic" employed alone or in combination with other
terms means, unless otherwise stated, means a non-aromatic cyclic
aliphatic group. Those skilled in the art will appreciate that alicyclic
groups include cycloalkyl and cycloalkenyl groups.

[0262] The term "heteroalicyclic" employed alone or in combination with
other terms means, unless otherwise stated, means an alicyclic group
wherein one or more of the carbon atoms in the ring are replaced with
heteroatoms independently selected from the group consisting of oxygen,
nitrogen, and sulfur. Those skilled in the art will appreciate that
heteroalicyclic groups include heterocyclyl groups. Examples of
heteroalicyclic groups include oxazolidinyl, piperidinyl, pyrrolidinyl
and tetrahydrofuranyl groups, and the like.

[0263] The term "amino" employed alone or in combination with other terms
means, unless stated otherwise, a --NR1R2 group, wherein
R1 and R2 are each independently selected from the group
consisting of hydrogen, C1-6 alkyl, and aryl.

[0264] The term "amido" employed alone or in combination with other terms
means, unless stated otherwise, an amino-C(O)-- group, wherein amino is
as defined herein.

[0265] The term "acylamino" employed alone or in combination with other
terms means, unless stated otherwise, a R1C(O)NR2-- group,
wherein R1 and R2 are each independently selected from the
group consisting of hydrogen, alkyl, and aryl.

[0266] The term "carboxyl" employed alone or in combination with other
terms means, unless stated otherwise, a R1C(O)O-- group, wherein
R1 is selected from the group consisting of hydrogen, alkyl, aryl,
and a metal cation.

[0267] The term "acyloxy" employed alone or in combination with other
terms means, unless stated otherwise, a R1C(O)O-- group, wherein
R1 is selected from the group consisting of hydrogen, alkyl, and
aryl.

[0268] The term "guanidino" employed alone or in combination with other
terms means, unless stated otherwise, an amino-C(NR1)NR2--
group, wherein amino is as defined herein and R1 and R2 are
each independently selected from the group consisting of hydrogen, alkyl,
and aryl.

[0269] The term "urea" employed alone or in combination with other terms
means, unless stated otherwise, an amino-C(O)--NR1-- group, wherein
amino is as defined herein and R1 is selected from the group
consisting of hydrogen, alkyl, and aryl.

[0270] The term "carbonate" employed alone or in combination with other
terms means, unless stated otherwise, a carboxyl-O-- group, wherein
carboxyl is as defined herein.

[0271] The term "thiourea" employed alone or in combination with other
terms means, unless stated otherwise, an amino-C(S)--NR1-- group,
wherein amino is as defined herein and R1 is selected from the group
consisting of hydrogen, alkyl, and aryl.

[0272] As used herein, the term "substituted" is intended to mean that one
or more hydrogen atoms in the group indicated is replaced with one or
more independently selected suitable substituents, provided that the
normal valency of each atom to which the substituent(s) are attached is
not exceeded, and that the substitution results in a stable compound.

[0273] The other general chemical terms used in the formulae herein have
their usual meanings.

[0274] Asymmetric centers exist in the phosphopeptide. The asymmetric
centers may be designated by the symbols R or S, depending on the
configuration of substituents in three dimensional space at the chiral
atom. All stereochemical isomeric forms of the compounds, including
diastereomeric, enantiomeric, and epimeric forms, as well as D-isomers
and L-isomers, erythro and threo isomers, syn and anti isomers, and
mixtures thereof are contemplated herein. In one embodiment, the
phosphopeptide comprises L-amino acids.

[0275] Individual enantiomers may be prepared synthetically from
commercially available enantiopure starting materials or by preparing an
enantiomeric mixture and resolving the mixture into individual
enantiomers. Resolution methods include conversion of the enantiomeric
mixture into a mixture of diastereomers and separation of the
diastereomers by, for example, recrystallization or chromatography;
direct separation of the enantiomers on chiral chromatographic columns;
or any other appropriate method known in the art. Starting materials of
defined stereochemistry may be commercially available or synthesised by
techniques known in the art. In one embodiment, the starting materials
include L-amino acids. In another embodiment, the starting materials
include natural L-amino acids.

[0276] Geometric isomers of the phosphopeptide may also exist. All cis,
trans, syn, anti, entgegen (E), and zusammen (Z) isomers, and mixtures
thereof are contemplated herein.

[0277] Tautomeric isomers of the phosphopeptide, for example, keto/enol
and imine/enamine tautomers, may also exist. All tautomeric isomers are
contemplated herein.

[0280] Base addition salts can be prepared by reacting a phosphopeptide
comprising a free acid with inorganic or organic bases. Examples of base
addition salts include: ammonium salts; alkali metal salts, for example
sodium salts and potassium salts; and alkaline earth metal salts, for
example calcium salts and magnesium salts. Other salts will be apparent
to those skilled in the art.

[0281] Quaternary salts of basic nitrogen-containing groups can be
prepared by reacting a phosphopeptide comprising a basic
nitrogen-containing group with, for example, alkyl halides, for example
methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;
dialkyl sulfates for example dimethyl, diethyl, dibutyl, and diamyl
sulfates; arylalkyl halides for example benzyl and phenylethyl bromides;
and the like. Other reagents suitable for preparing quaternary salts of
basic nitrogen-containing groups will be apparent to those skilled in the
art.

[0282] The phosphopeptide may form or exist as solvates with various
solvents. If the solvent is water, the solvate may be referred to as a
hydrate, for example, a mono-hydrate, a di-hydrate, or a tri-hydrate. All
solvated forms and unsolvated forms are contemplated herein.

[0283] Isotopologues and isotopomers of the phosphopeptide, wherein one or
more atoms in the phosphopeptide are replaced with different isotopes,
are also contemplated herein. Suitable isotopes include, for example,
1H, 2H (D), 3H (T), 12C, 13C, 14C,
16O, and 18O.

[0284] It is intended that reference to a range of numbers disclosed
herein (for example, 1 to 10) also incorporates reference to all rational
numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,
7, 8, 9 and 10) and also any range of rational numbers within that range
(for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all
sub-ranges of all ranges expressly disclosed herein are hereby expressly
disclosed. These are only examples of what is specifically intended and
all possible combinations of numerical values between the lowest value
and the highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.

[0285] Although the present invention is broadly as defined above, those
persons skilled in the art will appreciate that the invention is not
limited thereto and that the invention also includes embodiments of which
the following description gives examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0286] The invention will now be described with reference to the Figures
in which:

[0287] FIG. 1 is a transmission electron micrograph of iron nanoparticles
prepared by reducing iron (II) sulfate with sodium borohydride in the
absence of an additive;

[0288] FIG. 2 is a transmission electron micrograph of iron nanoparticles
prepared by reducing iron (II) sulfate with sodium borohydride in the
presence of sodium citrate;

[0289] FIGS. 3 and 4 are transmission electron micrographs of iron
nanoparticles prepared by reducing iron (II) sulfate with sodium
borohydride in the presence of 3-O-(phospho)-serine;

[0317] FIGS. 47, 48, and 49 are transmission electron micrographs of gold
nanoparticles prepared in the presence of phosphopeptide 209; and

[0318] FIG. 50 is an electron diffraction pattern of gold nanoparticles
prepared in the presence of phosphopeptide 209.

DETAILED DESCRIPTION OF THE INVENTION

[0319] The present invention provides a metal nanoparticle-phosphopeptide
complex comprising: [0320] a metal nanoparticle; and [0321] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the metal nanoparticle, [0322] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0323] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs.

[0324] In one embodiment, the metal nanoparticle is as defined in any of
the preceding embodiments.

[0325] In one embodiment, the size of the nanoparticle is from 3 to 250, 3
to 200, 3 to 150, 3 to 125, 3 to 100, 3 to 75, 3 to 50, 3 to 40, 3 to 30,
3 to 20, 3 to 10, 5 to 250, 5 to 200, 5 to 150, 5 to 125, 5 to 100, 5 to
75, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 7 to 250, 7 to 200, 7 to
150, 7 to 125, 7 to 100, 7 to 75, 7 to 50, 7 to 40, 7 to 30, 7 to 20, 7
to 10, 10 to 250, 10 to 200, 10 to 150, 10 to 125, 10 to 100, 10 to 75,
10 to 50, 10 to 40, 10 to 30, or 10 to 20 nm. In one embodiment, the size
of the nanoparticle is less than 250, 200, 150, 125, 100, 75, 50, 40, 30,
20, 10, 7, 5, or 3 nm.

[0326] In one embodiment, the metal nanoparticle is an iron nanoparticle.

[0327] In one embodiment, the iron nanoparticle is an iron oxide
nanoparticle. In one embodiment, the size of the iron oxide nanoparticle
is less than about 10 nm. In another embodiment, the iron oxide
nanoparticle is less than about 8 nm. In another embodiment, the size of
the iron oxide nanoparticle is about 5 nm. In one embodiment, the size of
the iron oxide nanoparticle is from about 5 nm to about 8 nm.

[0328] In another embodiment, the iron nanoparticle is an iron-iron oxide
core-shell nanoparticle. In one embodiment, the size of the iron-iron
oxide core-shell nanoparticle is less than about 50 nm. In another
embodiment, the size of the iron-iron oxide core-shell nanoparticle is
less than about 30 nm. In another embodiment, the size of the iron-iron
oxide core-shell nanoparticle is about 20 nm. In one embodiment, the size
of the iron-iron oxide core-shell nanoparticle is from about 8 nm to
about 50 nm. In another embodiment, the size of the iron-iron oxide
core-shell nanoparticle is from about 10 nm to about 50 nm. In another
embodiment, the size of the iron-iron oxide core-shell nanoparticle is
from about 8 nm to about 25 nm. In another embodiment, the size of the
iron-iron oxide core-shell nanoparticle is from about 15 nm to about 25
nm.

[0330] In one embodiment, the metal nanoparticle is a cobalt nanoparticle.

[0331] In one embodiment, the metal nanoparticle is a nickel nanoparticle.

[0332] In one embodiment, the metal nanoparticle is a copper nanoparticle.

[0333] In one embodiment, the metal nanoparticle is a ruthenium
nanoparticle. In one embodiment, the size of the ruthenium nanoparticle
is from about 20 nm to 100 nm.

[0334] In one embodiment, the metal nanoparticle is a rhodium
nanoparticle.

[0335] In one embodiment, the metal nanoparticle is a palladium
nanoparticle. In one embodiment, the size of the palladium nanoparticle
is from about 3 nm to about 7 nm. In another embodiment, the size of the
palladium nanoparticle is about 5 nm.

[0336] In one embodiment, the metal nanoparticle is a silver nanoparticle.

[0337] In one embodiment, the metal nanoparticle is an iridium
nanoparticle.

[0338] In one embodiment, the metal nanoparticle is a platinum
nanoparticle.

[0339] In one embodiment, the metal nanoparticle is a gold nanoparticle.
In one embodiment, the size of the gold nanoparticle is from about 3 nm
to about 5 nm. In another embodiment, the size of the gold nanoparticle
is about 4 nm.

[0340] In one embodiment, the metal nanoparticle exhibits
super-paramagnetic behaviour at room temperature. In another embodiment,
the metal nanoparticle exhibits ferrimagnetic behaviour at room
temperature. In another embodiment, the metal nanoparticle exhibits
ferromagnetic behaviour at room temperature.

[0341] A metal nanoparticle may comprise metals other than those
indicated, provided that the metallic composition of the nanoparticle is
not substantially altered. For example, in some embodiments an iron
nanoparticle may comprise, in addition to iron, one or more additional
metals, for example chromium, manganese, cobalt, nickel, copper, or zinc,
as minor components. In one embodiment, the metal indicated comprises
more than 70 mol % of the metal present in the metal nanoparticle. In
another embodiment, the metal indicated comprises more than 75 mol % of
the metal. In another embodiment, the metal indicated comprises more than
80 mol % of the metal. In another embodiment, the metal indicated
comprises more than 85 mol % of the metal. In another embodiment, the
metal indicated comprises more than 90 mol % of the metal. In another
embodiment, the metal indicated comprises more than 95 mol % of the
metal. In another embodiment, the metal indicated comprises more than 99
mol % of the metal.

[0342] The phosphopeptide of the metal nanoparticle-phosphopeptide complex
is on the surface of the metal nanoparticle.

[0343] The phosphopeptide complex comprises two or more
phosphorus-containing groups capable of interacting with the surface of
the metal nanoparticle. In one embodiment, the phosphorus-containing
groups interact with the surface of the metal nanoparticle. In one
embodiment, the phosphorus-containing groups interact strongly with the
surface of the metal nanoparticle. In one embodiment, the interaction is
of sufficient strength to prevent dissociation of the phosphopeptide from
the surface of the metal nanoparticle. In one embodiment, the
phosphopeptide is adsorbed on the surface of the iron nanoparticle.

[0344] Without wishing to be bound by theory, the applicant believes that
in some embodiments the phosphopeptide is adsorbed on the surface of the
metal nanoparticle by the interaction between the phosphorus-containing
groups and the surface of the metal nanoparticle.

[0345] The phosphopeptide comprises two or more contiguous peptide motifs.
Each peptide motif is bound directly to another peptide motif via a
peptide bond--i.e. the N-terminus of one peptide motif is bound to the
C-terminus of another peptide motif. For example, when the phosphopeptide
comprises three peptide motifs, the last amino acid of the first peptide
motif is bound directly via a peptide bond to the first amino acid of the
second peptide motif and the last amino acid of the second peptide motif
is bound directly via a peptide bond to the first amino acid of the third
peptide motif. The peptide backbone of each peptide motif in the
phosphopeptide is therefore bound so as to form a contiguous sequence of
amino acids.

[0346] Each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs.

[0347] In one embodiment, the phosphorus-containing group comprises a
phosphate, phosphonate, or pyrophosphate group. In one embodiment, the
phosphorus-containing group comprises a phosphate or phosphonate group.
In one embodiment, the phosphorus-containing group comprises a linker via
which a phosphate or phosphonate group is bound to the amino acid.

[0348] In one embodiment, a phosphate or phosphonate group is disposed at
the distal (with respect to the peptide backbone) end of the
phosphorus-containing group. In one embodiment, the phosphorus-containing
group is a phosphate or phosphonate group.

[0349] In one embodiment, the phosphopeptide comprises three or more
phosphorus-containing groups. In another embodiment, the phosphopeptide
comprises four or more phosphorus-containing groups.

[0350] In one embodiment, the phosphorus-containing groups are disposed on
the two or more contiguous peptide motifs at regular intervals. In one
embodiment, two or more phosphorus-containing groups are bound to amino
acids at the equivalent position in each peptide motif. For example, if
the first phosphorus-containing groups are bound to the first amino acid
of the first peptide motif bearing a phosphorus-containing group, then
the second and subsequent phosphorus groups will also be bound to the
first amino acid of any peptide motifs bearing phosphorus-containing
groups.

[0351] In one embodiment, each peptide motif contains one or more, two or
more, or three or more phosphorus-containing groups. In one embodiment,
each peptide motif contains one phosphorus-containing group. In one
embodiment, each peptide motif contains two phosphorus-containing groups.
In one embodiment, each peptide motif contains three
phosphorus-containing groups.

[0352] In one embodiment, each peptide motif contains one
phosphorus-containing group and the two or more phosphorus-containing
groups are bound to amino acids at the equivalent position in each
peptide motif.

[0353] Each peptide motif comprises a peptide backbone with the same
number of amino acids. In one embodiment, each peptide motif is 3 or more
amino acids in length. In another embodiment, each peptide motif is from
3 to 10 amino acids in length. In another embodiment, each peptide motif
is from 3 to 7 amino acids in length. In another embodiment, each peptide
motif is from 3 to 6 amino acids in length. In another embodiment, each
peptide motif is 3, 4, or 5 amino acids in length. In another embodiment,
each peptide motif is 3 amino acids in length.

[0354] In one embodiment, the phosphopeptide comprises 2 to 100 contiguous
peptide motifs. In another embodiment, the phosphopeptide comprises 2 to
50 contiguous peptide motifs. In another embodiment, the phosphopeptide
comprises 2 to 20 contiguous peptide motifs. In another embodiment, the
phosphopeptide comprises 2 to 10 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 4 contiguous peptide
motifs.

[0357] Many unnatural amino acids are commercially available. Those that
are not may be synthesised using standard methods known to those skilled
in the art.

[0358] In one embodiment, each chiral amino acid of the peptide motifs is
an L-amino acid. In one embodiment, the amino acids of the peptide motifs
comprise D-amino acids. In another embodiment, each chiral amino acid of
the peptide motif is a D-amino acid.

[0359] The amino acids at the equivalent position in each peptide motif
have similar structural and/or electronic properties. The phosphopeptide
therefore comprises a repeated sequence of amino acids having similar
structural and/or electronic properties.

[0360] A person skilled in the art will appreciate that when a
phosphorus-containing group is bound to an amino acid, the
phosphorus-containing group is to be excluded from the consideration of
whether the amino acid has structural and/or electronic properties
similar to amino acids at the equivalent position in other peptide
motifs. For example, a serine residue and an O-phospho-serine residue
(i.e. a serine residue substituted with a phosphorus-containing
group--the phospho group) meet the requirement of having similar
structural and/or electronic properties.

[0361] In one embodiment, the amino acids at the equivalent position in
each peptide motif have similar structural and electronic properties.

[0362] In one embodiment, the amino acids at the equivalent position in
each peptide motif have similar hydrophobicity/hydrophilicity. In another
embodiment, the amino acids at the equivalent position in each peptide
motif have similar polarity.

[0363] Natural amino acids can be classified, for example, by
hydrophobicity, hydrophilicity, and polarity. The same principles used to
classify natural amino acids can be used to classify unnatural amino
acids.

[0364] In one embodiment, the amino acids of each peptide motif are
selected from one of the following categories: polar amino acids, non
polar amino acids, hydrophobic amino acids, and non hydrophobic amino
acids; and the amino acids at the equivalent position in each peptide
motif are selected from the same category of amino acids.

[0367] In one embodiment, the charged amino acid is an acidic amino acid.
Acidic amino acids include, for example, aspartic acid and glutamic acid.
In one embodiment, the acidic amino acid is an aliphatic amino acid.
Acidic aliphatic amino acids include, for example, aspartic acid and
glutamic acid. In one embodiment, the acidic amino acid is a
heteroaliphatic amino acid. In another embodiment, the acidic amino acid
is an alicyclic amino acid. In another embodiment, the acidic amino acid
is a heteroalicyclic amino acid. In another embodiment, the acidic amino
acid is an aromatic amino acid. In another embodiment, the acidic amino
acid is a heteroaromatic amino acid.

[0368] In another embodiment, the charged amino acid is a basic amino
acid. Basic amino acids include, for example, arginine, histidine, and
lysine. In one embodiment, the basic amino acid is an aliphatic amino
acid. Basic aliphatic amino acids include, for example, arginine and
lysine. In one embodiment, the basic amino acid is a heteroaliphatic
amino acid. In another embodiment, the basic amino acid is an alicyclic
amino acid. In another embodiment, the basic amino acid is a
heteroalicyclic amino acid. In another embodiment, the basic amino acid
is an aromatic amino acid. In another embodiment, the basic amino acid is
a heteroaromatic amino acid. Basic heteroaromatic amino acids include,
for example, histidine.

[0369] In another embodiment, the polar amino acid is a neutral polar
amino acid. Neutral polar amino acids include, for example, serine,
threonine, asparagine, glutamine, cysteine, and tyrosine. In one
embodiment, the neutral polar amino acid is an aliphatic amino acid.
Neutral polar aliphatic amino acids include, for example, serine,
threonine, asparagine, glutamine, and cysteine. In one embodiment, the
neutral polar amino acid is a heteroaliphatic amino acid. In another
embodiment, the neutral polar amino acid is an alicyclic amino acid. In
another embodiment, the neutral polar amino acid is a heteroalicyclic
amino acid. In another embodiment, the neutral polar amino acid is an
aromatic amino acid. Neutral polar amino acids include, for example,
tyrosine. In another embodiment, the neutral polar amino acid is a
heteroaromatic amino acid. Neutral polar heteroaromatic acids include,
for example, [2-(triazolyl)methyl]glycine.

[0371] In another embodiment, the hydrophobic amino acid is an aliphatic
amino acid. Hydrophobic aliphatic amino acids include, for example,
valine, isoleucine, alanine, and leucine. In another embodiment, the
hydrophobic amino acid is an alicyclic amino acid. In another embodiment,
the hydrophobic amino acid is aromatic amino acid. Hydrophobic aromatic
amino acids include, for example, phenylalanine, tryptamine, and
tyrosine. In another embodiment, the hydrophobic amino acid is a
heteroaromatic amino acid. In another embodiment, the hydrophobic amino
acid is a heteroaliphatic amino acid. Hydrophobic heteroaliphatic amino
acids include, for example, methionine. In another embodiment, the
hydrophobic amino acid is a heteroalicyclic amino acid.

[0372] In another embodiment, the non-hydrophobic amino acid is a charged
amino acid as described herein.

[0374] In one embodiment, the amino acids at the equivalent position in
each peptide motif are substantially identical. In one embodiment, the
amino acids at the equivalent position in each peptide motif are
identical.

[0375] Without wishing to be bound by theory, the applicant believes that
the two or more contiguous peptide motifs, wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties may adopt a helical secondary structure in
solution.

[0376] In one embodiment, the phosphopeptide favours a helical structure
in solution. In one embodiment, the phosphopeptide has a helical
structure in solution.

[0377] In one embodiment, the amino acid sequence of the two or more
contiguous peptide motifs is such that the contiguous peptide motifs
favour a helical structure in solution. In one embodiment, the amino acid
sequence of the two or more contiguous peptide motifs is such that the
contiguous peptide motifs favour an amphipathic helical structure in
solution.

[0378] In one embodiment, the amino acid sequence of the phosphopeptide is
such that the phosphopeptide favours a helical structure in solution. In
one embodiment, the amino acid sequence of the phosphopeptide is such
that the phosphopeptide favours an amphipathic helical structure in
solution.

[0379] In one embodiment, the phosphorus-containing groups are presented
on the same side of the helical structure.

[0380] In an amphipathic helix, non-polar and/or hydrophobic amino acids
are predominantly on one side of the helix and polar and/or non
hydrophobic amino acids are predominantly on the other, resulting in a
peptide that is predominantly non-polar and/or hydrophobic on one face
and polar and/or non hydrophobic on the other. Certain amino acids are
known to favour the formation of a helical structure in solution, when
incorporated into a peptide. Examples include alanine, valine, leucine,
and phenylalanine. In one embodiment, each peptide motif comprises at
least one amino acid that favours the formation of a helical structure in
solution. Methods for determining the secondary structures of peptides in
solution are known in the art, for example, circular dichromism
spectroscopy.

[0381] In one embodiment, each peptide motif is a tripeptide. In one
embodiment, a phosphorus-containing group is optionally bound to the
first amino acid in each tripeptide motif. In another embodiment, a
phosphorus-containing group is optionally bound to the second amino acid
in each tripeptide motif. In another embodiment, a phosphorus-containing
group is optionally bound to the third amino acid in each tripeptide
motif.

[0382] In one embodiment, each peptide motif is a tripeptide, wherein: the
first amino acid of each peptide motif at each instance is independently
selected from one of the following categories: non polar amino acids and
hydrophobic amino acids; the second amino acid of each peptide motif at
each instance is independently selected from one of the following
categories: non polar amino acids, hydrophobic amino acids, polar amino
acids, and hydrophobic amino acids; the third amino acid of each peptide
motif at each instance is independently selected from one of the
following categories: polar amino acids and hydrophobic amino acids; a
phosphorus-containing group capable of interacting with the surface of
the metal nanoparticle is optionally bound to the third amino acid in
each peptide motif; and the amino acids at the equivalent position in
each peptide motif are selected from the same category of amino acids.

[0389] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from one of the following categories: non polar amino acids and
hydrophobic amino acids; the second amino acid of each peptide motif at
each instance is independently selected from one of the following
categories: non polar amino acids and hydrophobic amino acids; and the
third amino acid of each peptide motif at each instance is independently
selected from one of the following categories: polar amino acids and non
hydrophobic amino acids; a phosphorus-containing group capable of
interacting with the surface of the metal nanoparticle is optionally
bound to the third amino acid in each peptide motif; and the amino acids
at the equivalent position in each peptide motif are selected from the
same category of amino acids.

[0390] In one embodiment, each peptide motif is a tripeptide, wherein
[0391] the first amino acid of each peptide motif at each instance is
independently selected from one of the following categories: non polar
aliphatic amino acids, non polar heteroalicyclic amino acids, non polar
aromatic amino acids, hydrophobic aliphatic amino acids, and hydrophobic
heteroalicyclic amino acids; [0392] the second amino acid of each peptide
motif at each instance is independently selected from one of the
following categories: non polar aliphatic amino acids, non polar
heteroalicyclic amino acids, non polar aromatic amino acids, hydrophobic
aliphatic amino acids, and hydrophobic heteroalicyclic amino acids;
[0393] the third amino acid of each peptide motif at each instance is
independently selected from one of the following categories: polar
neutral aliphatic amino acids, polar neutral aromatic amino acids, non
hydrophobic neutral aliphatic amino acids, and non hydrophobic neutral
aromatic amino acids; [0394] a phosphorus-containing group capable of
interacting with the surface of the metal nanoparticle is optionally
bound to the third amino acid in each peptide motif; and [0395] the amino
acids at the equivalent position in each peptide motif are selected from
the same category of amino acids.

[0396] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine,
valine, proline, phenylalanine, and glycine; the second amino acid of
each peptide motif at each instance is independently selected from the
group consisting of alanine, isoleucine, leucine, valine, proline,
phenylalanine, and glycine; and the third amino acid of each peptide
motif optionally bound to a phosphorus-containing group at each instance
is independently selected from the group consisting of threonine,
O-phospho-threonine, serine, O-phospho-serine, tyrosine, and
O-phospho-tyrosine. In one embodiment, the phosphopeptide comprises from
2 to 50 contiguous peptide motifs. In one embodiment, the phosphopeptide
comprises from 2 to 20 contiguous peptide motifs.

[0397] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine,
valine, and proline; the second amino acid of each peptide motif at each
instance is independently selected from the group consisting of alanine,
isoleucine, leucine, valine, and proline; and the third amino acid of
each peptide motif optionally bound to a phosphorus-containing group at
each instance is independently selected from the group consisting of
threonine, O-phospho-threonine, serine, O-phospho-serine, tyrosine, and
O-phospho-tyrosine. In one embodiment, the phosphopeptide comprises from
2 to 50 contiguous peptide motifs. In one embodiment, the phosphopeptide
comprises from 2 to 20 contiguous peptide motifs.

[0398] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine, and
valine; the second amino acid of each peptide motif at each instance is
independently selected from the group consisting of alanine, isoleucine,
leucine, and valine; and the third amino acid of each peptide motif
optionally bound to a phosphorus-containing group at each instance is
independently selected from the group consisting of threonine,
O-phospho-threonine, serine, O-phospho-serine, tyrosine, and
O-phospho-tyrosine. In one embodiment, the phosphopeptide comprises from
2 to 50 contiguous peptide motifs. In one embodiment, the phosphopeptide
comprises from 2 to 20 contiguous peptide motifs.

[0399] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif is alanine; the second amino acid
of each peptide motif is alanine; and the third amino acid of each
peptide motif optionally bound to a phosphorus-containing group at each
instance is independently selected from the group consisting of threonine
or O-phospho-threonine; serine or O-phospho-serine; or tyrosine or
O-phospho-tyrosine. In one embodiment, the phosphopeptide comprises from
2 to 50 contiguous peptide motifs. In one embodiment, the phosphopeptide
comprises from 2 to 20 contiguous peptide motifs.

[0400] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from one of the following categories: non polar amino acids and
hydrophobic amino acids; the second amino acid of each peptide motif at
each instance is independently selected from one of the following
categories: non polar amino acids, hydrophobic amino acids, polar amino
acids, and hydrophobic amino acids; and the third amino acid of each
peptide motif at each instance is independently selected from one of the
following categories: polar amino acids and non hydrophobic amino acids;
a phosphorus-containing group capable of interacting with the surface of
the metal nanoparticle is optionally bound to the third amino acid in
each peptide motif; and the amino acids at the equivalent position in
each peptide motif are selected from the same category of amino acids.

[0407] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine,
valine, proline, phenylalanine, and glycine; the second amino acid of
each peptide motif at each instance is independently selected from the
group consisting of lysine, arginine, histidine, alanine, isoleucine,
leucine, valine, proline, phenylalanine, tryptamine, and glycine; and the
third amino acid of each peptide motif optionally bound to a
phosphorus-containing group at each instance is independently selected
from the group consisting of tyrosine, O-phospho-tyrosine, histidine,
phospho-histidine, [2-(triazolyl)-C1-6alkyl]glycine, and
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0408] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine,
valine, and proline; the second amino acid of each peptide motif at each
instance is independently selected from the group consisting of lysine
and arginine; and the third amino acid of each peptide motif optionally
bound to a phosphorus-containing group at each instance is independently
selected from the group consisting of tyrosine, O-phospho-tyrosine,
histidine, phospho-histidine, [2-(triazolyl)-C1-6alkyl]glycine, and
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0409] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine,
valine, and proline; the second amino acid of each peptide motif at each
instance is independently selected from the group consisting of alanine,
isoleucine, leucine, valine, and proline; and the third amino acid of
each peptide motif optionally bound to a phosphorus-containing group at
each instance is independently selected from the group consisting of
tyrosine, O-phospho-tyrosine, histidine, phospho-histidine,
[2-(triazolyl)-C1-6alkyl]glycine, and
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0410] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine, and
valine; the second amino acid of each peptide motif at each instance is
independently selected from the group consisting of lysine and arginine;
and the third amino acid of each peptide motif optionally bound to a
phosphorus-containing group at each instance is independently selected
from the group consisting of histidine, phospho-histidine,
[2-(triazolyl)-C1-6alkyl]glycine, and
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0411] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif at each instance is independently
selected from the group consisting of alanine, isoleucine, leucine, and
valine; the second amino acid of each peptide motif at each instance is
independently selected from the group consisting of alanine, isoleucine,
leucine, and valine; and the third amino acid of each peptide motif
optionally bound to a phosphorus-containing group at each instance is
independently selected from the group consisting of histidine,
phospho-histidine, [2-(triazolyl)-C1-6alkyl]glycine, and
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0412] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif is alanine; the second amino acid
of each peptide motif is alanine; and the third amino acid of each
peptide motif optionally bound to a phosphorus-containing group at each
instance is independently selected from the group consisting of threonine
and O-phospho-threonine. In one embodiment, the phosphopeptide comprises
from 2 to 50 contiguous peptide motifs. In one embodiment, the
phosphopeptide comprises from 2 to 20 contiguous peptide motifs.

[0413] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif is alanine; the second amino acid
of each peptide motif is alanine; and the third amino acid of each
peptide motif optionally bound to a phosphorus-containing group at each
instance is independently selected from the group consisting of serine
and O-phospho-serine. In one embodiment, the phosphopeptide comprises
from 2 to 50 contiguous peptide motifs. In one embodiment, the
phosphopeptide comprises from 2 to 20 contiguous peptide motifs.

[0414] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif is alanine; the second amino acid
of each peptide motif is alanine; and the third amino acid of each
peptide motif optionally bound to a phosphorus-containing group at each
instance is independently selected from the group consisting of tyrosine
and O-phospho-tyrosine. In one embodiment, the phosphopeptide comprises
from 2 to 50 contiguous peptide motifs. In one embodiment, the
phosphopeptide comprises from 2 to 20 contiguous peptide motifs.

[0415] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif is alanine; the second amino acid
of each peptide motif is lysine; and the third amino acid of each peptide
motif optionally bound to a phosphorus-containing group is
[2-(triazolyl)-C1-6alkyl]glycine or
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0416] In one embodiment, each peptide motif is a tripeptide, wherein the
first amino acid of each peptide motif is alanine; the second amino acid
of each peptide motif is alanine; and the third amino acid of each
peptide motif optionally bound to a phosphorus-containing group is
[2-(triazolyl)-C1-6alkyl]glycine or and
[2-(triazolyl)-C1-6alkyl]glycine wherein the triazolyl ring is
substituted with C1-6alkylphosphonate. In one embodiment, the
phosphopeptide comprises from 2 to 50 contiguous peptide motifs. In one
embodiment, the phosphopeptide comprises from 2 to 20 contiguous peptide
motifs.

[0417] In one embodiment, the [2-(triazolyl)-C1-6alkyl]glycine is
selected from the group consisting of [2-(triazolyl)-methyl]glycine,
[2-(2-[triazolyl]-ethyl)]glycine, and [2-(3-[triazolyl]-propyl)]glycine.
In another embodiment, the [2-(triazolyl)-C1-6alkyl]glycine is
[2-(triazolyl)-methyl]glycine.

[0418] In another embodiment, the [2-(triazolyl)-C1-6alkyl]glycine is
selected from the group consisting of [2-(triazol-4-yl)-methyl]glycine,
[2-(2-[triazol-4-yl]-ethyl)]glycine, and
[2-(3-[triazol-4-yl]-propyl)]glycine. In another embodiment, the
[2-(triazolyl)-C1-6alkyl]glycine is
[2-(triazol-4-yl)-methyl]glycine.

[0419] In another embodiment, the [2-(triazolyl)-C1-6alkyl]glycine is
selected from the group consisting of [2-(triazol-1-yl)-methyl]glycine,
[2-(2-[triazol-1-yl]-ethyl)]glycine, and
[2-(3-[triazol-1-yl]-propyl)]glycine. In another embodiment, the
[2-(triazol-1-yl)-C1-6alkyl]glycine is
[2-(triazol-1-yl)-methyl]glycine.

[0420] In one embodiment, the triazolyl in the
[2-(triazolyl)-C1-6alkyl]glycine is substituted with a
phosphorus-containing group. In one embodiment, the
[2-(triazolyl)-C1-6alkyl]glycine is
[2-(triazol-4-yl)-C1-6alkyl]glycine, wherein the 1-position of the
triazolyl ring is substituted with a phosphorus containing group. In
another embodiment, the [2-(triazolyl)-C1-6alkyl]glycine is
[2-(triazol-1-yl)-C1-6alkyl]glycine, wherein the 4-position of the
triazolyl ring is substituted with a phosphorus containing group.

[0421] In one embodiment, the phosphopeptide comprises one or more groups
that mitigate aggregation of the metal nanoparticle-phosphopeptide
complex with metal nanoparticles or other metal
nanoparticle-phosphopeptide complexes.

[0422] In one embodiment, the phosphopeptide is optionally substituted
with one or more groups that mitigate aggregation of the metal
nanoparticle-phosphopeptide complex with metal nanoparticles or other
metal nanoparticle-phosphopeptide complexes.

[0423] A person skilled in the art will appreciate that when a group that
mitigates aggregation is bound to an amino acid, the group that mitigates
aggregation is to be excluded from the consideration of whether the amino
acid has structural and/or electronic properties similar to amino acids
at the equivalent position in other peptide motifs. For example, a serine
residue and an O-sulfate-serine residue (i.e. a serine residue
substituted with a group that mitigates aggregation--the sulfate group)
meet the requirement of having similar structural and/or electronic
properties.

[0424] In one embodiment, the group that mitigates aggregation of the
metal nanoparticle-phosphopeptide complex with metal nanoparticles or
other metal nanoparticle-phosphopeptide complexes mitigates aggregation
by electrostatic stabilisation. In another embodiment, the group that
mitigates aggregation of the metal nanoparticle-phosphopeptide complex
with metal nanoparticles or other metal nanoparticle-phosphopeptide
complexes mitigates aggregation by steric stabilisation. In another
embodiment, the group that mitigates of the metal
nanoparticle-phosphopeptide complex with metal nanoparticles or other
metal nanoparticle-phosphopeptide complexes mitigates aggregation by
electrostatic stabilisation and steric stabilisation.

[0425] In one embodiment, the group that mitigates aggregation is a
charged group or a polymer.

[0426] In one embodiment, the group that mitigates aggregation comprises a
charged group. In one embodiment, the charged group is selected from the
group consisting of sulfate, C1-6alkylsulfate,
C2-6alkenylsulfate, C2-6alkynylsulfate, sulfonate,
C1-6alkylsulfonate, C2-6alkenylsulfonate, and
C2-6alkynylsulfonate. In one embodiment the group that mitigates
aggregation is selected from the group consisting of sulfate,
C1-6alkylsulfate, sulfonate, and C1-6alkylsulfonate.

[0427] In another embodiment, the group that mitigates aggregation is a
polymer. In one embodiment, the polymer is a short polymer. In one
embodiment, the polymer is a polymer selected from the group consisting
of polyethylene glycol, polyoxyethylene, polyethylene oxide, polyols,
polysaccharides, and any combination thereof. In another embodiment, the
polymer is a charged polymer. In one embodiment, the charged polymer is
selected from the group consisting of a charged peptide, poly(styrene
sulfonate), a zwitterionic polymer, and any combination thereof. In one
embodiment, the charged peptide is a sulfated peptide. In one embodiment,
the polymer is a synthetic polymer.

[0428] In another embodiment, the group that mitigates aggregation is a
peptide comprising one or more hydrophilic and/or polar amino acids. In
one embodiment, the peptide comprises from 2 to 20, from 2 to 19, from 2
to 18, from 2 to 17, from 2 to 16, from 2 to 15, from 2 to 14, from 2 to
13, from 2 to 12, from 2 to 11, from 2 to 10, from 2 to 9, from 2 to 8,
from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, or from 2 to 3 amino
acids. In one embodiment, the peptide is a tri-, tetra-, penta-, hexa-,
hepta-, octa-, nona-, deca-, undeca-, or dodeca-peptide. In one
embodiment, the one or more hydrophilic and/or polar amino acids are
charged amino acids. In one embodiment, the charged amino acid is
aspartic acid or glutamic acid. In one embodiment, the peptide comprises
or is a polyaspartic acid or polyglutamic acid sequence. In one
embodiment, the peptide is a hexa or deca-aspartic acid or glutamic acid
tag. In one embodiment, the peptide is attached to the two or more
contiguous peptide motifs via the C-terminus or N-terminus of the two or
more contiguous peptide motifs. In one embodiment, the peptide is
attached via the N-terminus of the two or more contiguous peptide motifs.

[0429] In one embodiment, the phosphopeptide further comprises one or more
groups that favour the formation of and/or stabilises a helical and/or
amphipathic secondary structure in solution. In one embodiment, the group
that favours the formation of and/or stabilises a helical and/or
amphipathic secondary structure in solution comprises a hydrogen bond
donor or acceptor. In one embodiment, the group that favours the
formation of and/or stabilises a helical and/or amphipathic secondary
structure in solution comprises an N-acetyl galactosamine residue.

[0430] In one embodiment, the phosphopeptide comprises an amino acid
sequence of the formula (I):

[0433] Xaa1, Xaa2, and Xaa3, respectively, at each instance
of n, have similar structural and/or electronic properties.

[0434] A person skilled in the art will appreciate that when a
phosphorus-containing group or a group that mitigates aggregation is
bound to R2, the optionally substituted ring formed when R1 and
R2 are taken together with nitrogen atom and carbon atom to which
they are attached, or the optionally substituted ring formed when R2
and R3 are taken together with the carbon atom to which they are
attached in a Xaa1, Xaa2, or Xaa3, the
phosphorus-containing group or group that mitigates aggregation is to be
excluded from the consideration of whether Xaa1, Xaa2, and
Xaa3, respectively, at each instance of n, have structural and/or
electronic properties. For example, if Xaa1 is a serine residue when
n is 1 and an O-phospho-serine residue when n is 2 (i.e. a serine residue
substituted with a phosphorus-containing group--the phospho group), these
two residues are considered to meet the requirement of having similar
structural and/or electronic properties.

[0435] In one embodiment, Xaa1, Xaa2, and Xaa3
respectively, at each instance of n have similar structural and
electronic properties.

[0436] In one embodiment, n is an integer from 2 to 50. In one embodiment,
n is an integer from 2 to 20. In another embodiment, n is an integer from
2 to 10. In another embodiment, n is an integer from 2 to 4.

[0437] In one embodiment, the phosphorus-containing group is selected from
the group consisting of phosphate, C1-6alkylphosphate,
C2-6alkenylphosphate, C2-6alkynylphosphate, arylphosphate,
C1-6alkylarylphosphate, C2-6alkenylarylphosphate,
C2-6alkynylarylphosphate, phosphonate, C1-6alkylphosphonate,
C2-6alkenylphosphonate, C2-6alkynylphosphonate,
arylphosphonate, C1-6alkylarylphosphonate,
C2-6alkenylarylphosphonate, C2-6alkynylarylphosphonate. In one
embodiment, the phosphorus-containing group is selected from the group
consisting of phosphate, phosphonate, C1-6alkylphosphate, and
C1-6alkylphosphonate.

[0438] The group that mitigates aggregation of the metal
nanoparticle-phosphopeptide complex with metal nanoparticles or other
metal nanoparticle-phosphopeptide complexes is as defined in any of the
embodiments described above.

[0439] In one embodiment, Xaa1 and Xaa2 are each independently
an amino acid residue of the formula (II) wherein: [0440] R1 is
selected from the group consisting of hydrogen, C1-6alkyl,
C2-6alkenyl, and C2-6alkynyl, each of which is optionally
substituted with one or more halo; [0441] R2 is selected from the
group consisting of hydrogen, C1-6alkyl, C2-6alkenyl,
C2-6alkynyl, and C1-6alkylaryl, each of which is optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
C1-6alkyoxy, C1-6alkylthio, halo, C1-6haloalkyl, and
C1-6haloalkoxy; [0442] R3 is selected from the group consisting
of hydrogen, C1-6alkyl, C2-6alkenyl, and C2-6alkynyl, each
of which is optionally substituted with one or more halo; [0443] or
R1 and R2 together with nitrogen atom and carbon atom to which
they are attached form a 5- or 6-membered heterocyclyl ring optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
C1-6alkyoxy, C1-6alkylthio, halo, C1-6haloalkyl, and
C1-6haloalkoxy; [0444] or R2 and R3 together with the
carbon atom to which they are attached form a 5- or 6-membered cycloalkyl
or cycloalkenyl optionally substituted with one or more substituents
independently selected from the group consisting of C1-6alkyl,
C2-6alkenyl, C2-6alkynyl, C1-6alkyoxy, C1-6alkylthio,
halo, C1-6haloalkyl, and C1-6haloalkoxy; and [0445] m is 0 or 1
and p is 0, or m is 0 and p is 0 or 1; or Xaa2 at each instance of n
is an amino acid residue of the formula (II) wherein: [0446] R1 is
selected from the group consisting of hydrogen, C1-6alkyl,
C2-6alkenyl, and C2-6alkynyl, each of which is optionally
substituted with one or more halo; [0447] R2 is selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
C1-6alkylaryl, and C1-6alkylheteroaryl, wherein each
C1-6alkyl, C2-6alkenyl, and C2-6alkynyl is substituted
with hydroxyl, thiol, amino, amido, carboxyl, or guanidino, and
optionally substituted with one or more substituents independently
selected from the group consisting of hydroxyl, C1-6alkyoxy, thiol,
C1-6alkylthio, halo, C1-6haloalkyl, C1-6haloalkoxy, cyano,
and nitro, each C1-6alkylaryl is substituted with hydroxyl, thiol,
or amino, and optionally substituted with one or more substituents
independently selected from the group consisting of C1-6alkyl,
C2-6alkenyl, C2-6alkynyl, hydroxyl, C1-6alkyoxy, thiol,
C1-6alkylthio, halo, C1-6haloalkyl, C1-6haloalkoxy, amino,
cyano, and nitro, and each C1-6alkylheteroaryl is optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy, amino, cyano, and nitro; [0448]
R3 is selected from the group consisting of hydrogen,
C1-6alkyl, C2-6alkenyl, and C2-6alkynyl, each of which is
optionally substituted with one or more halo; [0449] or R1 and
R2 together with nitrogen atom and carbon atom to which they are
attached form a 5- or 6-membered heterocyclyl ring substituted with
hydroxyl or thiol and optionally substituted with one or more
substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, cyano, and nitro; [0450] or R2 and R3
together with the carbon atom to which they are attached form a 5- or
6-membered cycloalkyl or cycloalkenyl ring substituted with hydroxyl or
thiol and optionally substituted with one or more substituents
independently selected from the group consisting of C1-6alkyl,
C2-6alkenyl, C2-6alkynyl, hydroxyl, C1-6alkyoxy, thiol,
C1-6alkylthio, halo, C1-6haloalkyl, C1-6haloalkoxy, or a
5- or 6-membered heterocyclyl ring optionally substituted with one or
more substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy; and [0451] m is 0 or 1 and p is 0, or m is 0 and p
is 0 or 1; and

[0452] Xaa3 at each instance of n is an amino acid residue of the
formula (II) wherein: [0453] R1 is selected from the group
consisting of hydrogen, C1-6alkyl, C2-6alkenyl, and
C2-6alkynyl, each of which is optionally substituted with one or
more halo; [0454] R2 is selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C1-6alkylaryl,
and C1-6alkylheteroaryl, wherein each C1-6alkyl,
C2-6alkenyl, and C2-6alkynyl is substituted with hydroxyl,
thiol, amino, amido, carboxyl, or guanidino, and optionally substituted
with one or more substituents independently selected from the group
consisting of hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio,
halo, C1-6haloalkyl, C1-6haloalkoxy, cyano, and nitro, each
C1-6alkylaryl is substituted with hydroxyl, thiol, or amino, and
optionally substituted with one or more substituents independently
selected from the group consisting of C1-6alkyl, C2-6alkenyl,
C2-6alkynyl, hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio,
halo, C1-6haloalkyl, C1-6haloalkoxy, amino, cyano, and nitro,
and each C1-6alkylheteroaryl is optionally substituted with one or
more substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, amino, cyano, and nitro; [0455] R3 is selected
from the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl,
and C2-6alkynyl, each of which is optionally substituted with one or
more halo; [0456] or R1 and R2 together with nitrogen atom and
carbon atom to which they are attached form a 5- or 6-membered
heterocyclyl ring substituted with hydroxyl or thiol and optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy, cyano, and nitro; [0457] or
R2 and R3 together with the carbon atom to which they are
attached form a 5- or 6-membered cycloalkyl or cycloalkenyl ring
substituted with hydroxyl or thiol and optionally substituted with one or
more substituents independently selected from the group consisting of
C1-6alkyl, C2-6alkenyl, C2-6alkynyl, hydroxyl,
C1-6alkyoxy, thiol, C1-6alkylthio, halo, C1-6haloalkyl,
C1-6haloalkoxy, or a 5- or 6-membered heterocyclyl ring optionally
substituted with one or more substituents independently selected from the
group consisting of C1-6alkyl, C2-6alkenyl, C2-6alkynyl,
hydroxyl, C1-6alkyoxy, thiol, C1-6alkylthio, halo,
C1-6haloalkyl, C1-6haloalkoxy; and [0458] m is 0 or 1 and p is
0, or m is 0 and p is 0 or 1; and a phosphorus-containing group is
optionally bound to R2, the optionally substituted ring formed when
R1 and R2 are taken together with nitrogen atom and carbon atom
to which they are attached, or the optionally substituted ring formed
when R2 and R3 are taken together with the carbon atom to which
they are attached in Xaa3; [0459] wherein the phosphorus-containing
group is a phosphate, phosphonate, C1-6alkylphosphate, or
C1-6alkylphosphonate.

[0460] In another embodiment, Xaa1 and Xaa2 are each
independently an amino acid residue of the formula (II) wherein: [0461]
R1 and R3 are each hydrogen; [0462] R2 is selected from
the group consisting of hydrogen, C1-6alkyl and C1-6alkylaryl,
wherein each C1-6alkyl is optionally substituted with
C1-6alkylthio; [0463] or R1 and R2 together with nitrogen
atom and carbon atom to which they are attached form a pyrrolidinyl ring;
and [0464] m is 0 and p is 0; or Xaa2 at each instance of n is an
amino acid residue of the formula (II) wherein: [0465] R1 and
R3 are each hydrogen; [0466] R2 is selected from the group
consisting of C1-6alkyl, C1-6alkylaryl, and
C1-6alkylheteroaryl, wherein each C1-6alkyl is substituted with
hydroxyl, thiol, amino, amido, carboxyl, or guanidino, and each
C1-6alkylaryl is substituted with hydroxyl; and [0467] m is 0 and p
is 0; and

[0468] Xaa3 at each instance of n is an amino acid residue of the
formula (II) wherein: [0469] R1 and R3 are each hydrogen;
[0470] R2 is selected from the group consisting of C1-6alkyl,
C1-6alkylaryl, and C1-6alkylheteroaryl, wherein each
C1-6alkyl is substituted with hydroxyl, thiol, amino, amido,
carboxyl, or guanidino, and each C1-6alkylaryl is substituted with
hydroxyl; [0471] R2 is optionally substituted with phosphate,
phosphonate, C1-6alkylphosphate, or C1-6alkylphosphonate; and
[0472] m is 0 and p is 0; and a phosphorus-containing group is optionally
bound to R2 in Xaa3; [0473] wherein the phosphorus-containing
group is a phosphate, phosphonate, C1-6alkylphosphate, or
C1-6alkylphosphonate.

[0474] In one embodiment, the phosphopeptide comprises an amino acid
sequence of the formula (III):

Xaa1-Xaa2-Xaa3-Xaa4n (III) [0475] wherein:
[0476] Xaa1, Xaa2, Xaa3, and n are as defined in any of
the embodiments recited above; and [0477] Xaa4 is an amino acid
residue of the formula (II) as defined in any of the embodiments
described above.

[0478] In another embodiment, the phosphopeptide comprises an amino acid
sequence of the formula (IV):

Xaa1-Xaa2-Xaa3-Xaa4-Xaa5n (IV) [0479]
wherein: [0480] Xaa1, Xaa2, Xaa3, and n are as defined in
any of the embodiments recited above; and [0481] Xaa4 and Xaa5
are each independently an amino acid residue of the formula (II) as
defined in any of the embodiments described above.

[0482] In another embodiment, the phosphopeptide comprises an amino acid
sequence of the formula (V):

Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6n (V)
[0483] wherein: [0484] Xaa1, Xaa2, Xaa3, and n are as
defined in any of the embodiments recited above; and [0485] Xaa4,
Xaa5, and Xaa6 are each independently an amino acid residue of
the formula (II) as defined in any of the embodiments described above.

[0486] The N-terminus and C-terminus of the amino acid sequences of the
formulae (I), (III), (IV), and (V) may be bound to any suitable
substituent, provided that the substituent does not adversely affect the
ability of the phosphopeptide to interact with the metal nanoparticle.

[0487] In one embodiment, a group that mitigates aggregation of the metal
nanoparticles as defined in any of the embodiments described herein is
attached to the N-terminus or C-terminus of the amino acid sequence. In
one embodiment, the group that mitigates aggregation is a charged
peptide. In one embodiment, the charged peptide comprises a polyaspartic
acid or polyglutamic acid sequence.

[0488] In one embodiment, the phosphopeptide is a compound of the formula
(VI):

A1 Xaa1-Xaa2-Xaa3nA2 (IV) [0489]
wherein: [0490] A1 is selected from the group consisting of
hydrogen, an amino acid, a peptide, and group that mitigates aggregation
of the metal nanoparticle-phosphopeptide complex with metal nanoparticles
or other metal nanoparticle-phosphopeptide complexes; [0491] A2 is
selected from the group consisting of hydroxyl, an amino acid, a peptide,
and group that mitigates aggregation of the metal
nanoparticle-phosphopeptide complex with metal nanoparticles or other
metal nanoparticle-phosphopeptide complexes; and [0492] Xaa1,
Xaa2, and Xaa3 are as defined in any of the embodiments
relating to the amino acid sequence of formula (I) described herein.

[0493] A1 is bound to the N-terminus of the amino acid sequence and
A2 is bound to the C-terminus of the amino acid sequence.

[0494] In one embodiment, A1 is selected from the group consisting of
hydrogen, an amino acid, and a peptide; and A2 is selected from the
group consisting of hydroxyl, an amino acid, and a peptide.

[0495] In one embodiment, the peptide comprises from 2 to 100 amino acid
residues. In another embodiment, the peptide comprises from 2 to 75 amino
acid residues. In another embodiment, the peptide comprises from 2 to 50
amino acid residues. In another embodiment the peptide comprises from 2
to 20 amino acid residues. In another embodiment, the peptide comprises
from 2 to 10 amino acid residues. In another embodiment, the peptide
comprises from 2 to 6 amino acid residues.

[0496] In one embodiment, the peptide mitigates aggregation of the metal
nanoparticles. In one embodiment, the peptide comprises one or more
hydrophilic and/or polar amino acids. In one embodiment, the peptide
comprises from 2 to 20, from 2 to 19, from 2 to 18, from 2 to 17, from 2
to 16, from 2 to 15, from 2 to 14, from 2 to 13, from 2 to 12, from 2 to
11, from 2 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6,
from 2 to 5, from 2 to 4, or from 2 to 3 amino acid residues. In one
embodiment, the peptide is a tri-, tetra-, penta-, hexa-, hepta-, octa-,
nona-, deca-, undeca-, or dodeca-peptide. In one embodiment, the
hydrophilic and/or polar amino acids are charged amino acids. In one
embodiment, the charged amino acid is aspartic acid or glutamic acid. In
one embodiment, the peptide comprises or is a polyaspartic acid or
polyglutamic acid sequence. In one embodiment, the peptide is a hexa- or
deca-aspartic acid or glutamic acid tag.

[0497] In one embodiment, A1 is a fatty acid ester. In another
embodiment, A1 is dodecanoyl.

[0498] In another embodiment, A1 is peptide. In one embodiment, the
peptide mitigates aggregation of the metal nanoparticles. In one
embodiment, the peptide is a charged peptide. In one embodiment, the
charged peptide comprises one or more aspartic acid or glutamic acid
residues. In one embodiment, the peptide comprises or is a polyaspartic
acid or polyglutamic acid tag. In one embodiment, the peptide is DDDDDD-,
wherein each D represents an aspartic acid residue. In another
embodiment, the peptide is EEEEEE- or EEEEEEEEEE-, wherein each E
represents a glutamic acid residue.

[0499] In one embodiment, the phosphopeptide is a compound of the formula
(VII):

A1 Xaa1-Xaa2-Xaa3-Xaa4nA2 (VII)
[0500] wherein: [0501] Xaa1, Xaa2, Xaa3, Xaa4, n,
A1, and A2 are as defined in any of the embodiments described
above.

[0502] In another embodiment, the phosphopeptide comprises an amino acid
sequence of the formula (IV):

A1 Xaa1-Xaa2-Xaa3-Xaa4-Xaa5nA2
(IV) [0503] wherein: [0504] Xaa1, Xaa2, Xaa3, Xaa4,
Xaa5, n, A1, and A2 are as defined in any of the
embodiments described above.

[0505] In another embodiment, the phosphopeptide comprises an amino acid
sequence of the formula (V):

A1 Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6nA2 (V) [0506] wherein: [0507] Xaa1, Xaa2,
Xaa3, Xaa4, Xaa5, Xaa6, n, A1, and A2 are
as defined in any of the embodiments described above.

[0508] In one embodiment, the phosphopeptide is selected from the group
consisting of:

[0509] Without wishing to be bound by theory, the applicant believes that
the phosphopeptides of the present invention form spherical cavities in
which metal nanoparticles are located. The phosphorus containing groups
capable of interacting with the surface of the metal nanoparticle are
oriented towards the center of the cavity. The side chains of the other
amino acid residues may be oriented away from the center of the cavity.
The side chains orientated away from the centre of the cavity may
mitigate aggregation, if they are of an appropriate nature (e.g.
hydrophobic, hydrophilic, etc.) having regard to the liquid reaction
medium (e.g. its polarity, pH, etc.).

[0510] In one embodiment, each peptide motif comprises at least one amino
acid that mitigates aggregation of metal nanoparticles or other metal
nanoparticle-phosphopeptide complexes in the liquid reaction medium.

[0511] Without wishing to be bound by theory, the applicant also believes
that the position of the phosphorus containing groups in the
phosphopeptide can control the size and shape of the metal nanoparticle.

[0512] In one embodiment, the iron nanoparticle-phosphopeptide complex
comprises more than one type of phosphopeptide--i.e. phosphopeptides of
different chemical structure.

[0513] In a further aspect, the present invention provides a
phosphopeptide as defined in any of the embodiments described herein.

[0514] The phosphopeptide may be prepared by any suitable method known in
the art. In one embodiment, amino acid sequence of the phosphopeptide is
prepared by solid phase synthesis.

[0515] Solid-phase phase synthesis is commonly used for the preparation of
peptides. Generally the procedure involves immobilising the first amino
acid of the peptide on a solid support, usually via a linker. Examples of
solid supports include Merrifield resin, ArgoGel® resin,
Tentagel® resin, PEG-PS resin, CLEAR® resin, PEGA resin, and the
like. A person skilled in the art will be able to select an appropriate
solid support without undue experimentation. The next amino acid of the
sequence, wherein the N-terminus of the amino acid is protected, is then
coupled. If the N-terminus of the solid phase bound amino acid is
protected, the protecting group will need to be removed prior to
coupling. Examples of common protecting groups include Fmoc
(9-fluorenylmethyloxycarbonyl) and Boc (tert-butyloxycarbonyl). Boc
groups can be removed using acids, for example, trifluoroacetic acid.
Fmoc groups can be removed using base, for example, piperidine. Examples
of suitable solvents for the deprotection reaction include, but are not
limited to, N,N-dimethylformamide, dimethylsulfoxide, dichloromethane,
acetonitrile, and mixtures thereof.

[0516] The coupling reaction is typically carried out in the presence of
one or more activating agents. Examples of activating agents include DCC,
DIC, HBTU, HATU, PyBOP, BOP, and the like. An agent that reduces the
racemisation, for example, HOBt or HOAt, can also be included. The
coupling reaction is carried out in any suitable solvent. Examples of
suitable solvent include, but are not limited to, N,N-dimethylformamide,
dimethylsulfoxide, dichloromethane, acetonitrile, water, and mixtures
thereof. The solid phase bound peptide is then washed to remove any
residual reagents from the coupling reaction, and then subjected to a
deprotection step.

[0517] The next amino acid of the sequence, wherein the N-terminus of the
amino acid is protected, is then coupled. The sequence is repeated as
necessary to prepare the desired peptide sequence. The peptide is then
cleaved from the solid phase support. The crude peptide is typically
purified. Purification is usually carried out by preparative HPLC.

[0519] The phosphopeptides of the present invention comprise two or more
phosphorus-containing groups capable of interacting with the surface of
the metal nanoparticle. Each phosphorus-containing group is bound to an
amino acid of the two or more contiguous peptide motifs. The phosphorus
containing groups may be introduced into the peptide by any suitable
method known in the art.

[0520] In one embodiment, the phosphorus-containing groups are introduced
into the peptide by coupling an amino acid comprising the
phosphorus-containing group to the peptide. For example, commercially
available Fmoc-Ser(HPO3Bn)-OH may be coupled with the peptide to
introduce a serine amino acid bearing a phosphate group. Those of skill
in the art will appreciate that the reaction conditions for subsequent
deprotection and coupling reactions may need to be modified to account
for the presence of the phosphorus-containing group in the peptide.

[0521] In another embodiment, the peptide may be reacted with a suitable
precursor of the phosphorus-containing group.

[0522] In one embodiment, the phosphorus-containing groups are introduced
using an azide-alkyne Huisgen cycloaddition `click` reaction. A peptide
comprising a α-propargyl glycine amino acid is reacted with
2-azidoethylphosphonic acid in the presence of a copper catalyst to from
a 1,2,3-triazole ring substituted with the phosphorus-containing group
(an ethylphosphonic acid group). Alternatively, a peptide comprising an
azido-amino acid is reacted with a propargyl phosphonic acid.

[0523] In another embodiment, the phosphorus-containing groups are
introduced using a nitrile-azide cycloaddition reaction. The
nitrile-azide cycloaddition reaction provides a tetrazole ring
substituted with the phosphorus containing group.

[0524] In a further aspect, the present invention provides a composition
comprising a plurality of metal nanoparticles and a phosphopeptide of the
present invention.

[0525] In a further aspect, the present invention provides a composition
comprising a plurality of metal nanoparticle-phosphopeptide complexes of
the present invention.

[0526] In one embodiment, the compositions further comprise a solvent in
which the meal nanoparticle-phosphopeptide complexes are suspended.
Advantageously, the metal nanoparticles and phosphopeptide of the present
invention or metal nanoparticle-phosphopeptide complexes of the present
invention form stable suspensions in suitable solvents, for example
water. In one embodiment, the suspension is stable for at least one day.

[0527] In another embodiment, the compositions are in the form of a
powder. The powder may be treated with a solvent to provide a suspension
of the metal nanoparticles and phosphopeptide of the present invention or
metal nanoparticle-phosphopeptide complexes. Advantageously, the metal
nanoparticle-phosphopeptide complexes of the present invention may
readily disperse when combined with suitable solvents, for example water,
to provide stable suspensions. In one embodiment, the suspension is
stable for at least one day. In one embodiment, the suspension is stable
for at least 2, 4, 6, 8, 12, 18, or 24 h.

[0528] In one embodiment, the compositions comprise a pharmaceutically
acceptable carrier, excipient, or diluent. Any suitable carrier,
excipient, or diluent known in the pharmaceutical arts may used. In one
embodiment the compositions are for use in the treatment of cancer. In
another embodiment, the compositions are for use as a contrast agent for
contrast enhancement in medical imaging.

[0529] In one embodiment, the compositions comprise more than one type of
metal nanoparticle-phosphopeptide complex. In another embodiment, the
compositions comprise more than one type of metal nanoparticles, for
example, iron-iron oxide core-shell nanoparticles and iron oxide
nanoparticles. In another embodiment, the compositions comprise more than
one type of phosphopeptide.

[0530] In a further aspect, the present invention provides a method for
preparing a metal nanoparticle-phosphopeptide complex, the method
comprising contacting [0531] a metal compound; and [0532] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the metal nanoparticle, [0533] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0534] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs; in a liquid reaction medium under conditions that form a metal
nanoparticle-phosphopeptide complex.

[0535] In a further aspect, the present invention provides a method for
preparing metal nanoparticles, the method comprising contacting [0536]
a metal compound; [0537] a phosphopeptide comprising two or more
contiguous peptide motifs and two or more phosphorus-containing groups
capable of interacting with the surface of the metal nanoparticle,
[0538] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0539]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs; and [0540] a reducing agent in a
liquid reaction medium to form metal nanoparticles.

[0541] In a further aspect, the present invention provides a method for
preparing a metal nanoparticle-phosphopeptide complex, the method
comprising contacting [0542] a metal compound; [0543] a phosphopeptide
comprising two or more contiguous peptide motifs and two or more
phosphorus-containing groups capable of interacting with the surface of
the metal nanoparticle, [0544] wherein the amino acids at the equivalent
position in each peptide motif have similar structural and/or electronic
properties, and [0545] wherein each phosphorus-containing group is bound
to an amino acid in the two or more contiguous peptide motifs; and
[0546] a reducing agent in a liquid reaction medium to form a metal
nanoparticle-phosphopeptide complex. [0547] The methods advantageously
provide a one-pot route for producing metal nanoparticles and metal
nanoparticle-phosphopeptide complexes. Any suitable metal compound may be
used.

[0548] The metal is reducible by a reducing agent in a liquid reaction
medium (i.e. in solution). In one embodiment, the metal compound is
reducible in a liquid reaction medium comprising water, an organic
solvent, or a mixture thereof. In one embodiment, the metal compound is
reducible in a liquid reaction medium comprising water. In another
embodiment, the metal compound is reducible in an aqueous liquid reaction
medium.

[0549] In one embodiment, the metal compound is at least partially soluble
in the liquid reaction medium. In one embodiment, the metal compound is
soluble.

[0550] The reaction is carried out under conditions conducive to formation
of the nanoparticles.

[0551] In one embodiment, the metal compound is a metal salt. Examples of
metal salts include but are not limited to metal chlorides, nitrates,
citrates, oxalates, sulfates, acetates, and the like. Other suitable
salts will be apparent to those skilled in the art.

[0554] In one embodiment, the iron compound is an iron (II) or (III)
compound. In another embodiment the iron compound is an iron (III)
compound. In another embodiment, the iron compound is an iron (II)
compound.

[0555] In one embodiment, the iron compound is an organo-iron compound.
Examples of organo-iron compounds include ferrocene and iron
pentacarbonyl.

[0556] In one embodiment, the iron compound is an iron salt. In one
embodiment, the iron salt is selected from the group consisting of iron
sulfates, iron acetoacetonates, iron oxalates, iron citrates, iron
ammonium sulfates, iron sulfates, iron chlorides, and iron nitrates. In
another embodiment, the iron salt is an iron (II) salt.

[0557] One embodiment utilises iron (II) sulfate.

[0558] The phosphopeptide affects the nucleation and growth of the metal
nanoparticles in the liquid reaction medium. The phosphopeptide is as
defined in any of the embodiments described herein.

[0559] In one embodiment, the molar concentration of phosphopeptide
relative to metal is low. In one embodiment, the molar concentration of
phosphopeptide relative to metal is less than about 25%. In another
embodiment, the molar concentration of phosphopeptide relative to metal
is less than about 15%. In one embodiment, the molar concentration of
phosphopeptide relative to metal is about 5%.

[0560] Without wishing to be bound by theory, the applicant believes that
the phosphopeptides slow the rate of growth of the metal nanoparticles,
either by adsorbing onto the growing surface of the nanoparticles or by
reducing the quantity of metal compound available, resulting in smaller
nanoparticles.

[0561] The metal compound is reduced by the reducing agent in the presence
of the phosphopeptide to provide the metal nanoparticle-phosphopeptide
complex.

[0562] In one embodiment, the metal nanoparticle is an iron nanoparticle.
In one embodiment, the iron nanoparticle is an iron-iron oxide core-shell
nanoparticle. In one embodiment, the size of the iron-iron oxide
core-shell nanoparticle is less than about 50 nm. In another embodiment,
the size of the iron-iron oxide core-shell nanoparticle is less than
about 30 nm. In another embodiment, the size of the iron-iron oxide
core-shell nanoparticle is about 20 nm. In one embodiment, the size of
the iron-iron oxide core-shell nanoparticle is from about 10 nm to about
50 nm. In one embodiment, the size of the iron-iron oxide core-shell
nanoparticle is from about 8 nm to about 50 nm. In another embodiment,
the size of the iron-iron oxide core-shell nanoparticle is from about 15
nm to about 25 nm. In one embodiment, the size of the iron-iron oxide
core-shell nanoparticle is from about 8 nm to about 25 nm.

[0563] In one embodiment, the shell of the iron-iron oxide core-shell
nanoparticle is about 5 nm.

[0564] In another embodiment, the iron nanoparticle is an iron oxide
nanoparticle. In one embodiment, the size of the iron oxide nanoparticle
is less than about 10 nm. In another embodiment, the iron oxide
nanoparticle is about 8 nm.

[0565] In one embodiment, the metal nanoparticle is a cobalt nanoparticle.

[0566] In one embodiment, the metal nanoparticle is a nickel nanoparticle.

[0567] In one embodiment, the metal nanoparticle is a copper nanoparticle.

[0568] In one embodiment, the metal nanoparticle is a ruthenium
nanoparticle. In one embodiment, the size of the ruthenium nanoparticle
is from about 20 nm to 100 nm.

[0569] In one embodiment, the metal nanoparticle is a rhodium
nanoparticle.

[0570] In one embodiment, the metal nanoparticle is a palladium
nanoparticle. In one embodiment, the size of the palladium nanoparticle
is from about 3 nm to about 7 nm. In another embodiment, the size of the
palladium nanoparticle is about 5 nm.

[0571] In one embodiment, the metal nanoparticle is a silver nanoparticle.

[0572] In one embodiment, the metal nanoparticle is an iridium
nanoparticle.

[0573] In one embodiment, the metal nanoparticle is a platinum
nanoparticle.

[0574] In one embodiment, the metal nanoparticle is a gold nanoparticle.
In one embodiment, the size of the gold nanoparticle is from about 3 nm
to about 5 nm. In another embodiment, the size of the gold nanoparticle
is about 4 nm.

[0575] In one embodiment, the metal nanoparticles produced by the methods
have a relatively narrow size distribution. In one embodiment, the
standard deviation of the particle size is less than the mean particle
size. In another embodiment, the metal nanoparticles are substantially
monodisperse.

[0576] The reducing agent is selected having regard to the nature of the
metal compound. Examples of suitable reducing agents include but are not
limited to citrate, hydrazine, bitartrate, carbon monoxide, ascorbic
acid, hydrogen, metal hydrides, and the like.

[0578] In one embodiment, the metal hydride is a metal borohydride. In one
embodiment, the metal borohydride is selected from the group consisting
of lithium borohydride, sodium borohydride, sodium cyanoborohydride,
potassium borohydride, and lithium triethylborohydride. In one
embodiment, the metal borohydride is sodium borohydride.

[0579] In one embodiment, the liquid reaction medium comprises a solvent.
In one embodiment, the solvent is selected from the group consisting of
aqueous solvents, organic solvents, and mixtures thereof. Organic
solvents include, but are not limited to, dimethyl formamide;
dimethylsulfoxide; alcohols, for example methanol, ethanol, iso-propanol,
and tert-butanol; ethers, for example tetrahydrofuran and diethyl ether;
acetonitrile; nitromethane; chlorinated solvents, for example
dichloromethane, chloroform, and carbon tetrachloride; aromatic solvents,
for example benzene; and esters, for example ethyl acetate.

[0580] Advantageously, the methods can be carried out using non-toxic,
aqueous or water miscible solvent systems.

[0581] In one embodiment, the solvent is an aqueous solution. In one
embodiment, the aqueous solution is water.

[0583] The metal compound, reducing agent, and phosphopeptide may be
contacted at any suitable temperature. In one embodiment, the metal
compound, reducing agent, and phosphopeptide are contacted at ambient
temperature. In another embodiment, the metal compound, reducing agent,
and phosphopeptide are contacted at elevated temperature. In one
embodiment, the elevated temperature is less than 200° C.

[0584] In one embodiment, the contacting step is carried out under an
atmosphere of inert gas. In one embodiment, the inert gas is nitrogen or
argon. Carrying out the reaction under an atmosphere of inert gas
prevents the metal nanoparticles from being oxidising while they are
growing. The metal nanoparticles may be oxidised on exposure to air.

[0585] Relatively small nanoparticles may be completely oxidised on
exposure to air, while relatively large nanoparticles may only be
partially oxidised. Oxidation of relatively large metal nanoparticles may
result in the formation of metal-metal oxide core-shell nanoparticles.

[0586] In one embodiment, the methods further comprise mixing the metal
compound, phosphopeptide, and reducing agent in the liquid reaction
medium. Mixing the metal compound, phosphopeptide, and reducing agent in
the liquid reaction medium ensures that the reaction mixture is
homogeneous. The reaction mixture may be mixed by any method known in the
art.

[0587] In one embodiment, the methods further comprise recovering the
product metal nanoparticles or metal nanoparticle-phosphopeptide complex.
Suitable methods include, but are not limited to, filtration,
centrifugation, decanting, and magnetic separation. In one embodiment,
the metal nanoparticles or metal nanoparticle-phosphopeptide complex is
recovered by magnetic separation.

[0588] The recovered metal nanoparticles or metal
nanoparticle-phosphopeptide complex may be further purified by, for
example, washing the nanoparticles or nanoparticle-phosphopeptide complex
in a suitable solvent. Suitable solvents include, but are not limited,
water, ethanol, dimethyl sulfoxide, and dimethyl formamide.

[0589] The product metal nanoparticles or metal
nanoparticle-phosphopeptide complexes may be stored in the form of
powder. Conveniently, the powder may readily disperse when treated with a
solvent to provide a stable suspension of the metal nanoparticles or
metal nanoparticle-phosphopeptide complex in solution. Alternatively, the
product metal nanoparticles or metal nanoparticle-phosphopeptide
complexes may be stored in the form a suspension in a suitable solvent.
Suitable solvents include, but are not limited to, water and ethanol.

[0590] In one embodiment, the metal nanoparticle exhibits
super-paramagnetic behaviour at room temperature. In another embodiment,
the metal nanoparticle exhibits ferrimagnetic behaviour at room
temperature. In another embodiment, the metal nanoparticle exhibits
ferromagnetic behaviour at room temperature.

[0591] In a further aspect, the present invention provides metal
nanoparticles prepared by a method of the present invention.

[0592] In a further aspect, the present invention provides metal
nanoparticle-phosphopeptide complex prepared by a method of the present
invention.

[0593] In one embodiment, the metal nanoparticle is an iron nanoparticle.
In one embodiment, the iron nanoparticles exhibit ferromagnetic,
ferromagnetic, or superparamagnetic behaviour at room temperature.

[0594] In a further aspect, the present invention provides a method for
preparing a metal nanoparticle-phosphopeptide complex, the method
comprising contacting [0595] a metal compound; and [0596] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the metal nanoparticle, [0597] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0598] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs; and in a liquid reaction medium under conditions that precipitate
a metal nanoparticle-phosphopeptide complex.

[0599] Various methods are available for forming metal nanoparitcles in
solution by precipitation. Examples include, but are not limited to,
formation of insoluble hydroxides, oxides, or sulfides, precipitation by
addition of solvents in which the metal compound is insoluble or only
sparingly soluble, and irradiation. A person skilled in the art will be
able to select appropriate conditions having regard to the nature of the
metal compound.

[0600] In one embodiment, the metal is as defined in any of the preceding
embodiments.

[0601] In one embodiment, the method comprises contacting two or more
metal compounds. In one embodiment, at least two of the two or more metal
compounds comprise different metals.

[0602] In one embodiment, the method comprises co-precipitating two or
more metal compounds in the presence of the phosphopeptide to form the
metal nanoparticle phosphopeptide complex.

[0603] In one embodiment, at least one of the metal compounds comprises
iron. Examples of metals suitable for co-precipitation with iron include
but are not limited to cobalt and nickel.

[0604] In one embodiment, the metal compound is a metal salt. In one
embodiment, the metal salt is as defined in any of the preceding
embodiments.

[0605] In one embodiment, the metal nanoparticle is a metal oxide, metal
hydroxide, or metal chalcogenide nanoparticle.

[0606] In one embodiment, the method comprises contacting one or more
metal compounds, the phosphopeptide, and hydroxide or chalcogen ions. In
one embodiment, the chalcogen ions are anions. In one embodiment, the
chalcogen is sulfur. In one embodiment, sulfur anions are provided in the
form of hydrogen sulfide.

[0607] In one embodiment, the conditions comprise a base.

[0608] In one embodiment, the metal nanoparticles are iron nanoparticles.

[0609] In a further aspect, the present invention provides a method for
preparing iron nanoparticles, the method comprising contacting [0610]
iron (II); [0611] iron (III); [0612] a phosphopeptide comprising two or
more contiguous peptide motifs and two or more phosphorus-containing
groups capable of interacting with the surface of the iron nanoparticle,
[0613] wherein the amino acids at the equivalent position in each peptide
motif have similar structural and/or electronic properties, and [0614]
wherein each phosphorus-containing group is bound to an amino acid in the
two or more contiguous peptide motifs; and [0615] a base in a liquid
reaction medium to form iron nanoparticles.

[0616] In a further aspect, the present invention provides a method for
preparing an iron nanoparticle-phosphopeptide complex, the method
comprising contacting [0617] iron (II); [0618] iron (III); [0619] a
phosphopeptide comprising two or more contiguous peptide motifs and two
or more phosphorus-containing groups capable of interacting with the
surface of the iron nanoparticle, [0620] wherein the amino acids at the
equivalent position in each peptide motif have similar structural and/or
electronic properties, and [0621] wherein each phosphorus-containing
group is bound to an amino acid in the two or more contiguous peptide
motifs; and [0622] a base in a liquid reaction medium to provide an
iron nanoparticle-phosphopeptide complex.

[0624] In one embodiment, the metal is as defined in any of the preceding
embodiments.

[0625] In one embodiment, the metal is iron.

[0626] In one embodiment, iron (II) is formed in situ by the reduction of
an iron (III) compound with a reducing agent, for example hydrogen or
sodium borohydride. In another embodiment, iron (III) is formed in situ
by the oxidation of an iron (II) compound with an oxidising agent, for
example nitrate or oxygen.

[0627] In one embodiment, iron (II) is provided to the liquid reaction
mixture in the form of an iron (II) compound and the iron (III) is
provided to the liquid reaction mixture in the form of an iron (III)
compound.

[0628] In one embodiment, the iron (II) compound is an iron (II) salt. In
another embodiment, the iron (III) compound is an iron (III) salt. In one
embodiment, the iron salts are selected from the group consisting of iron
sulfates, iron acetoacetonates, iron oxalates, iron citrates, iron
ammonium sulfates, iron sulfates, iron chlorides, and iron nitrates.

[0630] In one embodiment, the ratio of iron (II) to iron (III) is about
1:2.

[0631] The phosphopeptide affects the nucleation and growth of the metal
nanoparticles in the liquid reaction medium. The phosphopeptide is as
defined in any of the embodiments described herein.

[0632] Without wishing to be bound by theory, the applicant believes that
the phosphopeptides slow the rate of growth of the metal nanoparticles,
either by adsorbing onto the growing surface of the nanoparticles or by
reducing the quantity of metal compound available, resulting in smaller
nanoparticles.

[0633] In one embodiment, the method is for preparing iron nanoparticles
or an iron nanoparticle-phosphopeptide complex. In one embodiment, the
molar ratio of phosphorus-containing groups to iron is less than 1:1. In
another embodiment, the molar ratio of phosphorus-containing groups to
iron is from about 0.05:1 to about 0.95:1. In another embodiment, the
molar ratio of phosphorus-containing groups to iron is from about 0.05:1
to about 0.75:1. In another embodiment, the molar ratio of
phosphorus-containing groups to iron is from about 0.05:1 to about 0.5:1.
In another embodiment, the molar ratio of phosphorus-containing groups to
iron is from about 0.1:1 to about 0.4:1.

[0634] In one embodiment, the molar ratio of phosphorus-containing groups
to iron (III) is less than 1:1. In another embodiment, the molar ratio of
phosphorus-containing groups to iron (III) is from about 0.05:1 to about
0.95:1. In another embodiment, the molar ratio of phosphorus-containing
groups to iron (III) is from about 0.05:1 to about 0.75:1. In another
embodiment, the molar ratio of phosphorus-containing groups to iron (III)
is from about 0.05:1 to about 0.5:1. In another embodiment, the molar
ratio of phosphorus-containing groups to iron (III) is from about 0.1:1
to about 0.4:1.

[0635] The iron (II) and iron (III) coprecipitate in the presence of the
base and the phosphopeptide to provide the iron nanoparticles or iron
nanoparticle-phosphopeptide complex.

[0636] In one embodiment, the iron nanoparticle is an iron oxide
nanoparticle. In one embodiment, the size of the iron oxide nanoparticle
is less than about 10 nm. In another embodiment, the iron oxide
nanoparticle is less than about 8 nm. In another embodiment, the iron
oxide nanoparticle is about 5 nm.

[0637] In one embodiment, the metal nanoparticles produced by the methods
have a relatively narrow size distribution. In one embodiment, the
standard deviation of the particle size is less than the mean particle
size. In another embodiment, the metal nanoparticles are substantially
monodisperse.

[0638] Any suitable base may be used in the methods. In one embodiment,
the base is ammonia. In another embodiment, the base is sodium hydroxide.
In another embodiment, the base is an organic base. In one embodiment,
the organic base is an organic amine. Examples of suitable organic amines
include, but are not limited to, triethylamine, diisopropylethylamine

[0639] A person skilled in the art will appreciate that the
coprecipitation reaction may be sensitive to pH and will be able to
select appropriate bases for particular metal compounds.

[0641] In one embodiment, the liquid reaction medium comprises a solvent.
In one embodiment, the solvent is selected from the group consisting of
aqueous solvents, organic solvents, and mixtures thereof. Organic
solvents include, but are not limited to, dimethyl formamide;
dimethylsulfoxide; alcohols, for example methanol, ethanol, iso-propanol,
and tert-butanol; ethers, for example tetrahydrofuran and diethyl ether;
acetonitrile; nitromethane; chlorinated solvents, for example
dichloromethane, chloroform, and carbon tetrachloride; aromatic solvents,
for example benzene; and esters, for example ethyl acetate.

[0642] Advantageously, the methods can be carried out using non-toxic,
aqueous or water miscible solvent systems.

[0643] In one embodiment, the solvent is an aqueous solution. In one
embodiment, the aqueous solution is water.

[0644] In one embodiment, the liquid reaction medium is water.

[0645] In one embodiment, the liquid reaction medium further comprises a
buffer. A person skilled in the art will be able select an appropriate
buffer considering the nature of components present in the liquid
reaction medium and the desired pH, without undue experimentation.

[0646] In one embodiment, the contacting step is carried out at ambient
temperature. In one embodiment, the contacting step is carried out under
an atmosphere of inert gas. In one embodiment, the inert gas is nitrogen
or argon. Carrying out the reaction under an atmosphere of inert gas
prevents the metal nanoparticles from being oxidising while they are
growing. The metal nanoparticles may be oxidised upon exposure to air.

[0647] In one embodiment, the methods further comprise mixing the metal
compound, phosphopeptide, and base in the liquid reaction medium. Mixing
the metal compound, phosphopeptide, and base in the liquid reaction
medium ensures that the reaction mixture is homogeneous. The reaction
mixture may be mixed by any method known in the art.

[0648] In one embodiment, the methods further comprise recovering the
product metal nanoparticles or metal nanoparticle-phosphopeptide complex.
Suitable methods include, but are not limited to, filtration,
centrifugation, and decanting. In one embodiment, the metal nanoparticles
or metal nanoparticle-phosphopeptide complex is recovered by magnetic
separation.

[0649] The recovered metal nanoparticles or metal
nanoparticle-phosphopeptide complex may be further purified by, for
example, washing the nanoparticles or nanoparticle-phosphopeptide complex
in a suitable solvent. Suitable solvents include, but are not limited,
water, ethanol, and dimethyl sulfoxide, dimethyl formamide.

[0650] The product metal nanoparticles or metal
nanoparticle-phosphopeptide complexes may be stored in the form of
powder. Conveniently, the powder may readily disperse when treated with a
solvent to provide a stable suspension of the metal nanoparticles or
metal nanoparticle-phosphopeptide complex in solution. Suitable solvents
include, but are not limited to, water, ethanol, dimethyl sulfoxide, and
dimethyl formamide.

[0651] In one embodiment, the metal nanoparticle exhibits
super-paramagnetic behaviour at room temperature. In another embodiment,
the metal nanoparticle exhibits ferrimagnetic behaviour at room
temperature. In another embodiment, the metal nanoparticle exhibits
ferromagnetic behaviour at room temperature.

[0652] In one embodiment, the metal nanoparticles are iron nanoparticles.
In one embodiment, the nanoparticles exhibit ferromagnetic,
ferrimagnetic, or superparamagnetic behaviour at room temperature.

[0653] In a further aspect the present invention provides an metal
nanoparticles prepared by a method of the present invention.

[0654] In a further aspect the present invention provides an metal
nanoparticle-phosphopeptide complex prepared by a method of the present
invention.

[0655] Advantageously, the metal nanoparticles in the metal
nanoparticle-phosphopeptide complex prepared according to the methods of
the present invention may exhibit superparamagnetic, ferromagnetic,
and/or ferrimagnetic properties.

[0656] The metal nanoparticles and metal nanoparticle-phosphopeptide
complexes of the present invention may be useful in medical applications,
for example, the treatment of cancer by hyperthermia and as agents for
contrast enhancement in medical imaging.

[0657] A person skilled in the art will be able to determine suitable
doses of the metal nanoparticles or metal nanoparticle-phosphopeptide
complex without undue experimentation.

[0658] The metal nanoparticles or metal nanoparticle-phosphopeptide
complex may be formulated for administration by any method known in the
art. Advantageously, the metal nanoparticles are and metal
nanoparticle-phosphopeptide complex of the present invention may be
capable of forming stable suspension in aqueous solutions.

[0659] Formulation of the metal nanoparticles or metal
nanoparticle-phosphopeptide complex for use in medical applications, for
example as a drug or contrast agent, can include binding an antibody to
the nanoparticles. The presence of the phosphopeptides on the surface of
the metal nanoparticles in the metal nanoparticle-phosphopeptide complex
is a particular advantage for such procedures since the phosphopeptides
can offer a range of chemical functionality that can be used for such
binding. Methods for binding antibodies are well known in the art.
EDC-NHS coupling of amino groups to carboxylic acid groups is one such
method.

[0660] Formulation of the metal nanoparticles or metal
nanoparticle-phosphopeptide complex for use in medical applications, for
example as a drug or contrast agent, can also include binding compounds
designed to minimise non-specific interactions of the particle with the
surfaces of cells of the body, or to minimise inflammatory reactions.
Such compounds are well-known in the art and include materials such as
poly(ethyleneoxide), otherwise known as PEG. For example,
amino-terminated PEG can be bound to chemical functionalities in the
phosphopeptides of the nanoparticle-phosphopeptide complex using EDC-NHS
coupling.

[0661] The present invention also provides various kits for preparing
agents for use in treating cancer and in medical imaging as defined
above.

[0662] The metal compound, phosphopeptide, and metal
nanoparticle-phosphopeptide complex are as defined in any of the
preceding embodiments.

[0663] Compounds for minimising non-specific interactions or inflammatory
reactions will be apparent to the skilled worker. The specific compound
used may depend on the intended application. Suitable compounds include,
for example, PEG molecules.

[0664] Examples of targeting groups include antibodies, antibody
fragments, singe chain antibodies, peptides, nucleic acids,
carbohydrates, lipids, lectins, drugs, and any other compounds that bind
to specifically targets in vivo. Other targeting groups will be apparent
to those skilled in the art.

[0665] The coupling reagent, if necessary, depends on the nature of the
compound and/or targeting group to be coupled and the specific reaction
involved. For peptide couplings, numerous activating agents are
commercially available.

[0666] The methods of the present invention for preparing metal
nanoparticle-phosphopeptide complexes may conveniently provide metal
nanoparticle-phosphopeptide complexes in a form suitable for
administration without purification. For example, the preparation of iron
nanoparticle-phosphopeptide complexes by reduction of iron (II) sulphate
with sodium borohydride in water in the presence of a phosphopeptide
rapidly provides an aqueous suspension of iron
nanoparticle-phosphopeptide complexes and non-toxic by products (sodium
borate). The iron nanoparticle-phosphopeptide complex can readily be
coupled to, for example, various antibodies using standard techniques.

[0667] In a further aspect, the present invention provides a metal
nanoparticle-phosphopeptide complex of the present invention for use as a
catalyst.

[0668] In a further aspect, the present invention provides a catalyst
comprising a metal nanoparticle-phosphopeptide complex of the present
invention.

[0669] Advantageously, the metal nanoparticles of the present invention
have significantly greater surface area than, for example, bulk metal.
The increased surface area may provide enhanced activity in the catalysis
of various desirable chemical reactions. The metal nanoparticles used
depend on the reaction to be catalysed. Examples of possible catalyst
applications include but are not limited to air cathodes in fuel cells,
oxidation catalysts (e.g. for the conversion of CO to CO2 in vehicle
exhausts), and partial oxidation catalysts in various industrial
reactions (e.g. for creating syngas).

[0670] The catalyst metal nanoparticles may be coated onto a support for
use. Examples of suitable supports include but are not limited to
ceramics and carbon (including both monolithic and particulate forms
thereof).

[0671] The metal nanoparticle-phosphopeptide complexes have numerous other
applications, as would be appreciated by a person skilled in the art. For
example, silver metal nanoparticles may be useful as antimicrobial agents
and metal chalcogenide nanoparticles may be useful as quantum dots
(semiconducting nanoparticles with band gaps that are particle size and
shape dependent and therefore tunable).

[0672] A person skilled in the art will appreciate that the optimum size
of the metal nanoparticles in the metal nanoparticle in the present
invention may vary depending on the intended application.

[0673] The following non-limiting examples are provided to illustrate the
present invention and in no way limit the scope thereof.

EXAMPLES

General Information

[0674] All reagents were purchased as reagent grade and used without
further purification. Solvents were used as supplied or dried according
to standard protocols (Perrin, D. D. et al., Purification of Laboratory
Chemicals, Pergamon Press Ltd., Oxford, 2nd Ed., 1980). The progress
of reactions was monitored by analytical thin layer chromatography (TLC)
using 0.2 mm thick pre-coated silica gel plates (Merck Kieselgel 60
F254 or Riedel-de Haen Kieselgel S F254). Compounds were
visualized by ultra-violet fluorescence or by staining with potassium
permanganate solution, followed by heating the plate, as appropriate.
Separation of mixtures was performed by flash chromatography using Merck
Kieselgel 60 (230-400 mesh) with the indicated solvents Infrared spectra
were obtained on an FTIR spectrometer as neat samples and absorption
maxima are expressed in wavenumbers (cm-1). 1H NMR spectra were
recorded on a Bruker AC 300 (300 MHz) spectrometer at ambient
temperature. Chemical shifts are expressed in parts per million downfield
from tetramethylsilane as an internal standard, and are reported as
chemical shift (δ in ppm), relative integral, multiplicity,
coupling constant (J in Hz) and assignment. 13C NMR spectra were
recorded on a Bruker AC 300 (75 MHz) spectrometer at ambient temperature
with complete proton decoupling. Electrospray ionization (ESI) mass
spectra were recorded using a Thermo Finnigan Surveyor MSQPlus
spectrometer, a Bruker micrOTOF-Q II spectrometer, or a hp Series 1100
MSD spectrometer. Transmission electron microscopy (TEM) images, electron
diffraction patterns and energy dispersive spectroscopy (EDS) data were
acquired digitally with a JEOL 2010 operated at an accelerating voltage
of 200 KeV and equipped with an Oxford Inca EDS detector. The samples for
TEM studies were prepared by resuspending the dry particles in ethanol
using sonication, depositing a few drops of ethanol suspension on a
copper or carbon-coated copper TEM grid, and allowing the ethanol to
evaporate under ambient conditions. Magnetisation measurements were
carried out on a superconducting quantum interference device (SQUID)
magnetometer or a Quantum Design physical property measurement system
(PPMS) using the Model P525 vibrating sample magnetometer (VSM)
measurement system at 0 and 300 K. For the SQUID device, dry particles
were weighted into a gelatin capsule which was then sealed and inserted
in the SQUID sample holder for measurement. Dynamic light scattering
measurements were carried out using a Malvern Zetasizer Nano ZS.

[0677] Solid phase peptide synthesis was performed using a Liberty
Microwave Peptide Synthesizer (CEM Corporation, Mathews, N.C.), using the
Fmoc/tBu strategy. Fmoc-Ala-Wang-Polystyrene resin was used as the
starting material. Peptide synthesis was carried out at 0.1 mmol scale.
Fmoc-Thr(HPO3Bn)-OH was used for the coupling of phosphorylated
threonine.

[0678] The Fmoc group was deprotected with 20% v/v piperidine in DMF for
30 seconds followed by a second deprotection for three minutes using a
microwave power of 60 W for both deprotections. The maximum temperature
for both deprotections was set at 75° C. Once a phosphorylated
threonine was introduced in the peptide chain, the Fmoc deprotection was
performed in the absence of microwave irradiation for 5 min and repeated
for 15 min without microwave. The coupling steps were performed with 5
equivalents of Fmoc protected amino acid in DMF (0.2 M), 4.5 equivalents
of HBTU in DMF (0.45 M) and 10 equivalents of NMM in DMF (2 M). Standard
amino acid couplings were performed for five minutes at 25 W at a maximum
temperature of 75° C. Fmoc-Thr(HPO3Bn)-OH couplings were
performed for 15 min at 25 W with a maximum temperature of 72° C.
The amino acid immediately following the phosphorylated residue was also
coupled for 15 min at 25 W with a maximum temperature of 72° C.
Boc-Ala-OH was used as the last residue and coupled for five minutes at
25 W at a maximum temperature of 75° C.

[0679] Following completion of the sequence, the peptide was released from
the resin with concomitant removal of protecting groups by treatment with
TFA/TIPS/H2O (95/2.5/2.5, v/v/v) at room temperature for three to
five hours as required. The crude peptide was precipitated with cold
diethyl ether, isolated by centrifugation, washed with cold diethyl
ether, dissolved in 1:1 (v/v) acetonitrile:water containing 0.1% TFA and
lyophilized.

[0680] The crude peptide product was analyzed for purity by analytical
RP-HPLC (Dionex P680) at 210 and 254 nm using a Gemini C18
(4.60×250 mm, 110A, 50 column (Phenomenex) at 1 mL/min. The solvent
system used was A (0.1% TFA in H2O) and B (0.1% TFA in MeCN). Final
purification was performed using Water 600 RP-HPLC using a Gemini C18
(10.00×250 mm, 110A, 50 column (Phenomenex). The solvent system
used was A (0.1% TFA in H2O) and B (0.1% TFA in MeCN). Final purity
was determined by analytical RP-HPLC (Dionex P680) using the same
conditions as for the crude product. Peptide mass was confirmed by LC-MS
(Dionex Ultimate 3000 equipped with a Thermo Finnigan Surveyor MSQPlus
spectrometer) using ESI in the positive mode: AATpAATpAATpAATpAA, wherein
Tp=phosphorylated threonine, (2.9 mg, 2.0%):
C42H76N14O31P4; MW=1453.20 g.mol-1; m/z
(ESI) 1453.4 [M+H].sup.+; 727.2 [M+2H]2+.

Phosphopeptide 106

##STR00013##

[0682] Solid phase peptide synthesis was performed using a Tribute Peptide
Synthesizer (Protein Technologies, Inc), using an Fmoc/tBu strategy.
Fmoc-Ala-Wang-Polystyrene resin was used as the starting material.
Peptide synthesis was carried out at 0.1 mmol scale.
Fmoc-Tyr(HPO3Bn)-OH was used for the coupling of phosphorylated
tyrosine.

[0683] The Fmoc group was deprotected with 20% v/v piperidine in DMF for 5
min followed by a second deprotection for 15 min at room temperature. The
coupling steps were performed with 5 equivalents of Fmoc protected amino
acid in DMF (0.25 M), 4.5 equivalents of HBTU in DMF (0.24 M) and 10
equivalents of NMM in DMF (2 M). Standard amino acid couplings were
performed for 40 min at room temperature. Fmoc-Tyr(HPO3Bn)-OH
couplings were performed for 1 h at room temperature. The amino acid
immediately following each phosphorylated amino acid was also coupled for
1 h at room temperature. Fmoc-Ala-OH was used as the last residue and
coupled for 40 min at room temperature.

[0684] The peptide was deprotected and released from the resin, and the
crude peptide isolated, analyzed, and purified as described above for the
synthesis of phosphopeptide 105: AAYpAAYpAAYpAAYpAA, wherein
Yp=phosphorylated tyrosine, (2.7 mg, 1.6%):
C42H76N14O31P4; MW=1701.48 g.mol-1; m/z
(ESI) 1702.3 [M+H].sup.+; 851.2 [M+2H]2+.

[0687] Phosphopeptide 107 was also prepared using a procedure analogous to
that described above for the preparation of phosphopeptide 106, using
Fmoc-Ser(HPO3Bn)-OH instead of Fmoc-Tyr(HPO3Bn)-OH and using
Boc-Ala-OH as the last amino acid instead of Fmoc-Ala-OH: (6.0 mg, 4.3%):
C42H76N14O31P4; MW=1397.2 g.mol-1; m/z
(ESI) 1397.34 [M+H].sup.+; 699.20 [M+2H]2+.

[0689] Peptide synthesis was carried out at 0.1 mmol scale.
Fmoc-Ser(HPO3Bn)-OH was used for the coupling of phosphorylated
serine.

[0690] The Fmoc group was deprotected with 20% v/v piperidine in DMF for 5
min followed by a second deprotection for 15 min at room temperature. The
coupling steps were performed with 5 equivalents of Fmoc protected amino
acid in DMF (0.25 M), 4.5 equivalents of HBTU in DMF (0.24 M) and 10
equivalents of NMM in DMF (2 M). Standard amino acid couplings were
performed for 40 min at room temperature. Fmoc-Ser(HPO3Bn)-OH
couplings were performed for 1 h at room temperature. The amino acid
immediately following each phosphorylated amino acid was also coupled for
1 h at room temperature. Boc-Ala-OH was used as the last residue and
coupled for 40 min at room temperature.

[0691] The peptide was deprotected and released from the resin, and the
crude peptide isolated, analyzed, and purified as described above for the
synthesis of phosphopeptide 105: (9.9 mg, 7.1%):
C42H76N14O31P4; MW=1397.2 g.mol-1; m/z
(ESI) 1397.3 [M+H].sup.+; 699.2 [M+2H]2+.

Phosphopeptide 108

##STR00015##

[0693] Solid phase peptide synthesis was performed using a Tribute Peptide
Synthesizer (Protein Technologies, Inc), using an Fmoc/tBu strategy.
Fmoc-Ala-Wang-Tentagel resin was used as the starting material. Peptide
synthesis was carried out at 0.1 mmol scale. Fmoc-Ser(HPO3Bn)-OH was
used for the coupling of phosphorylated serine.

[0694] The Fmoc group was deprotected with 20% v/v piperidine in DMF for 5
min followed by a second deprotection for 15 min at room temperature. The
coupling steps were performed with 5 equivalents of Fmoc protected amino
acid in DMF (0.25 M), 4.5 equivalents of HBTU in DMF (0.24 M) and 10
equivalents of NMM in DMF (2 M). Standard amino acid couplings were
performed for 40 min at room temperature. Fmoc-Ser(HPO3Bn)-OH
couplings were performed for 1 h at room temperature. The amino acid
immediately following each phosphorylated amino acid was also coupled for
1 h at room temperature. Boc-Ala-OH was used as the last residue and
coupled for 40 min at room temperature.

[0695] The peptide was deprotected and released from the resin, and the
crude peptide isolated, analyzed, and purified as described above for the
synthesis of phosphopeptide 105: AASpAASpAASAASAA, wherein
Sp=phosphorylated serine, (12.0 mg, 9.7%):
C42H74N14O25P2; MW=1237.06 g.mol-1; m/z
(ESI) 1237.44 [M+H].sup.+; 619.22 [M+2H]2+.

[0700] Commercially available Fmoc-Ser(HPO3Bn)-OH (200 mg, 0.4 mmol)
was dissolved in a mixture of 20% diethylamine in DMF (2 mL) and stirred
for two hours at room temperature. The solvent was then removed under
reduced pressure. The mixture was then dissolved in methanol (2 mL), 10%
Pd/C (10 mg) was added and the reaction was left to stir overnight with
H2 gas bubbling into the mixture. The suspension was then filtered
through on Celite® and the solvent evaporated under reduced pressure.
After suspension in water (3 mL), the suspension was washed with diethyl
ether (2×3 mL) and ethyl acetate (2×3 mL). The aqueous phase
was collected and the water evaporated under reduced pressure. The
resulting residue was recrystallised from a mixture of water, cold
diethyl ether and methanol to afford 112 (44 mg, 60%) as a white powder.
IR vmax (neat) 3002 (br), 1629, 1516, 1088, 987, 760; 111 NMR (300
MHz, D2O) δ 4.18-4.06 (2H, m, H(3), 3.94-3.91 (1H, m,
Hβ); m/z (ESI) 184.00 [M-H].sup.-, 369.01 [M2-H].sup.-. The
1H NMR, IR and MS data obtained were in agreement with that reported
in the literature (Arnold, L. D. et al. J. Am. Chem. Soc. 1988, 110,
2237-2241).

Peptide 113

##STR00018##

[0702] Solid phase peptide synthesis was performed using a Liberty
Microwave Peptide Synthesizer (CEM Corporation, Mathews, N.C.), using the
Fmoc/tBu strategy. Fmoc-Ala-Wang-Polystyrene resin was used as the
starting material. Peptide synthesis was carried out at 0.1 mmol scale.

[0703] The Fmoc group was deprotected with 20% v/v piperidine in DMF for
30 seconds followed by a second deprotection for three minutes using a
microwave power of 60 W for both deprotections. The maximum temperature
for both deprotections was set at 75° C. The coupling steps were
performed with 5 equivalents of Fmoc protected amino acid in DMF (0.2 M),
4.5 equivalents of HBTU in DMF (0.45 M) and 10 equivalents of DIPEA in
N-methylpyrrolidone (2 M) Amino acid couplings were performed for 5 min
at 25 W at a maximum temperature of 75° C.

[0704] The peptide was deprotected and released from the resin, and the
crude peptide isolated.

[0705] Final purification was performed using RP-HPLC (Water 600LCD) using
a Jupiter C18 (10.00×250 mm, 300 Å, 5μ) column (Phenomenex).
The solvent system used was A (0.1% TFA in H2O) and B (0.1% TFA in
MeCN). Final purity was determined by analytical RP-HPLC (Dionex P680) at
210 and 254 nm using an Aqua C18 (4.60×250 mm, 125 Å, 5μ)
column (Phenomenex) at 1 mL/min using a linear gradient. The solvent
system used was A (0.1% TFA in H2O) and B (0.1% TFA in MeCN).
Peptide mass was confirmed by LC-MS (Dionex Ultimate 3000 equipped with a
Thermo Finnigan Surveyor MSQPlus spectrometer) using ESI in the positive
mode: AATAATAATA, (21.0 mg, 26%); C33H58N10O14;
MW=818.9 g.mol-1; m/z (ESI) 819.4 [M+H].sup.+; 1638.3 [M+2H]2+.

Peptide 77

##STR00019##

[0707] Peptide 77 was prepared using a procedure analogous to that
described above for the preparation of peptide 113.

[0708] Following completion of the sequence the peptide was released from
the resin with concomitant removal of protecting groups by treatment with
TFA/TIPS/H2O (95/2.5/2.5, v/v/v) either at room temperature for two
to three hours as required or under microwave irradiation for 18 min at
10 W with a maximum temperature of 35° C. The crude peptide was
precipitated with cold diethyl ether, isolated by centrifugation, washed
with cold diethyl ether, dissolved in 1:1 (v/v) acetonitrile:water
containing 0.1% TFA and lyophilised.

[0709] The crude peptide was analysed for purity by analytical RP-HPLC
(Dionex P680) at 210 and 254 nm using an Aqua C18 (4.60×250 mm, 125
Å, 5μ) column (Phenomenex) at 1 mL/min using a linear gradient of
1% to 95% B over 30 min. The solvent system used was A (0.1% TFA in
H2O) and B (0.1% TFA in MeCN). Final purification was performed
using RP-HPLC (either Water 600LCD or Gilson 281) using a Jupiter C18
(10.00×250 mm, 300 Å, 50 column (Phenomenex) at 5 ml/min using
a linear gradient. The solvent system used was A (0.1% TFA in H2O)
and B (0.1% TFA in MeCN). Final purity was determined by analytical
RP-HPLC (Dionex P680) at 210 and 254 nm using an Aqua C18 (4.60×250
mm, 125 Å, 50 column (Phenomenex) at 1 mL/min using a linear
gradient. Again, the solvent system used was A (0.1% TFA in H2O) and
B (0.1% TFA in MeCN). Peptide mass was confirmed by LC-MS (Dionex
Ultimate 3000 equipped with a Thermo Finnigan Surveyor MSQPlus
spectrometer) using ESI in the positive mode: AATAATPATAATPA, (37 mg,
31%):

[0712] Diethyl 2-azidoethylphosphonate (370 mg, 1.8 mmol) was dissolved in
DMF (2.5 mL) and the solution was cooled to -5° C.
Trimethylbromosilane (12.5 mL, 7.1 mmol) was then added and the solution
left to stir overnight at room temperature. The solvent was evaporated
and co-evaporated with dry toluene (3×5 mL). The residue was
redissolved in water (10 mL), stirred overnight at room temperature then
concentrated and concentrated with water (3×10 mL). The residue was
then dissolved in water and freeze-dried to afford the title compound
(200 mg, 74%) as an oil. No further purification was carried out and the
crude product was used directly for the click reaction onto the peptide.
IR vmax (neat) 2778 (br), 2098, 1115, 927; 1H NMR (400 MHz,
D2O) δ 1.85 (2H, dt, J=18.1 Hz, J=7.1 Hz, CH2), 3.30 (2H,
dt, J=15.7 Hz, J=7.1 Hz, CH2); m/z (ESI) 150.14 [M-H].sup.-, 301.29
[M2-H].sup.-. The 1H NMR and MS data obtained were in agreement
with that reported in the literature (Alexandrova, L. A. et al. Nucleic
Acids Res. 1998, 26, 778-786).

Synthesis of Phosphopeptide 111

(i) Peptide 117

##STR00020##

[0714] Peptide 117 was prepared using a procedure analogous to that
described above for the preparation of phosphopeptide 105, using
Fmoc-Lys-Wang-Polystyrene resin as the starting material instead of
Fmoc-Ala-Wang-Polystyrene resin and using Fmoc-Gly(Propagyl)-OH instead
of Fmoc-Thr (HPO3Bn)-OH: AKPraAKPraAKPraAKPraAK, wherein
Pra=propargyl glycine.

[0715] Peptide 117 was also prepared by the following method.

[0716] Solid phase peptide synthesis based on Fmoc protection strategy was
performed on a 0.1 mmol scale using aminomethylated polystyrene resin
(Mitchell, A. R. et al., J. Org. Chem., 1978, 43(14), 2845-2852) (loading
1.0 mmol/g). The aminomethylated resin was swollen in DCM (5 mL) for 15
min and then the solvent was drained.
Fmoc-L-Lys(Boc)-OCH2PhOCH2CH2CO2H (2 eq) was
dissolved in 1 mL of DCM, DIC (2 eq) was added and the reaction mixture
was added to resin followed by agitating for 1 h. The mixture was drained
and the resin was washed with DMF (3×) and DCM (3×).

[0717] The peptide chain was assembled by manual SPPS.

[0718] N.sup.α-Protected amino acids Fmoc-Ala-OH and
Fmoc-Lys(Boc)-OH (5 eq) were dissolved in 2 mL of 0.23 M HBTU/DMF (4.6
eq), 0.5 ml of 2M NMM/DMF (10 eq) were added and the mixture was
transferred to the reaction vessel. The mixture was shaken for 45 min,
filtered and washed with DMF (3×) and DCM (3×). The
N.sup.α-protecting group was removed by 20% piperidine solution in
DMF (3 mL, 2×5 min), filtered and washed with DMF (3×) and
DCM (3×). The N.sup.α-protecting group was removed by 20%
piperidine solution in DMF (3 mL, 2×5 min), filtered and washed
with DMF (3×) and DCM (3×).

[0725] Solid phase peptide synthesis based on Fmoc protection strategy was
performed on a 0.1 mmol scale using aminomethylated polystyrene resin
(Mitchell, A. R. et al., J. Org. Chem., 1978, 43(14), 2845-2852) (loading
1.0 mmol/g). The aminomethylated resin was swollen in DCM (5 mL) for 15
min and then the solvent was drained.
Fmoc-L-Ala-OCH2PhOCH2CH2CO2H (2 eq) was dissolved in
1 mL of DCM, DIC (2 eq) was added and the reaction mixture was added to
resin followed by agitating for 1 h. The mixture was drained and the
resin was washed with DMF (3×) and DCM (3×).

[0726] Residues AKSAKSA of the peptide chain were assembled by automated
SPPS using a Tribute® peptide synthesizer and the Fmoc/tBu strategy.

[0727] N.sup.α-Protected amino acids Fmoc-Ala-OH and
Fmoc-Lys(Boc)-OH (5 eq) were dissolved in 2 mL of 0.23 M HBTU/DMF (4.6
eq), 0.5 ml of 2M NMM/DMF (10 eq) were added and the mixture was
transferred to the reaction vessel. The mixture was shaken for 45 min,
filtered and washed with DMF (3×) and DCM (3×). The
N.sup.α-protecting group was removed by 20% piperidine solution in
DMF (3 mL, 2×5 min), filtered and washed with DMF (3×) and
DCM (3×).

[0729] Residues AAPraAAPra of the peptide chain were assembled by manual
SPPS using the Fmoc/tBu strategy.

[0730] Couplings of N.sup.α-Fmoc-protected amino acids (5 eq) were
carried out in 45 min at room temperature in the presence of HBTU (4.6
eq) and NMM (10 eq) in DMF. The N.sup.α-protecting group was
removed by 20% piperidine solution in DMF (3 mL, 2×5 min).

[0731] Following completion of the sequence, the peptide was cleaved from
the resin with concomitant removal of the protecting groups by treating
the resin with 100 μL TIPS, 250 μL H2O, 250 μL
3,6-dioxa-1,8-octanedithiol and 9.4 mL TFA and agitating the mixture for
2 h at room temperature. The TFA solution was filtered and the peptide
was precipitated by addition of hexane/diethyl ether (1:1). After
centrifugation and washing with hexane/diethyl ether (1:1) the peptide
was lyophilised from 0.1% trifluoroacetic acid-water to yield 122.0 mg of
crude AAPraAAPraAKSAKSAA. m/z (ESI-MS): [M+2H.sup.+] calculated
mass=604.6, observed mass=604.8; [M+3H.sup.+] calculated mass=403.4,
observed mass=403.4.

[0761] Peptides 300-303 were prepared by the following general procedure.

[0762] Solid phase peptide synthesis based on Fmoc/tBu strategy was
performed on a 0.1 mmol scale using aminomethylated polystyrene resin or
TentaGel HL-NH2 resin derivatised with
Fmoc-L-Ala-OCH2Ph-OCH2CH2CO2H or
Fmoc-L-Lys(Boc)-OCH2PhOCH2CH2CO2H.

[0763] The peptide chains were assembled using either manual Fmoc SPPS or
a Tribute® peptide synthesiser as described for peptide 200, standard
amino acids or the building blocks Fmoc-L-propargylglycine and
Fmoc-Ser(HPO3Bn)-OH(Sp).

[0765] Peptides 300-302 were carried through to the next step without
further purification. Final purification and characterisation of peptide
303 was performed using RP-HPLC and LC-MS.

[0766] Semi-preparative RP-HPLC was performed as described for peptide
111. Analytical RP-HPLC was performed as described for peptide 111, using
the Phenomenex Gemini C18, 3 μm, 4.6 mm×150 mm column at a
flow rate of 1 mL/min.

[0770] Peptide 301 was prepared using a procedure analogous to that
described for peptide 200, using
Fmoc-L-Lys(Boc)-OCH2PhOCH2CH2CO2H instead of
Fmoc-L-Ala-OCH2PhOCH2CH2CO2H, and using only manual
SPPS to assemble the peptide chain. Stearic acid was attached as
described above. Lyophilisation yielded 134.2 mg of crude stearic
acid-AKpraAKpraAK. m/z (ESI-MS): [M+2H.sup.+] calculated mass=536.7,
observed mass=537.4.

Peptide 302

##STR00036##

[0772] Peptide 302 was prepared using a procedure analogous to that
described above for the preparation of peptide 200, using
Fmoc-L-Lys(Boc)-OCH2PhOCH2CH2CO2H, instead of
Fmoc-L-Ala-OCH2PhOCH2CH2CO2H and using manual SPPS to
assemble residues AKPraAKPraA of the peptide chain and automated SPPS to
assemble residues RRRRRR. Lyophilisation yielded 81.4 mg of crude
RRRRRRAKpraAKpraAA. m/z (ESI-MS):

[0775] Peptide 303 was prepared using a procedure analogous to that
described above for the preparation of peptide 200, using
Fmoc-L-Lys(Boc)-OCH2PhOCH2CH2CO2H, instead of
Fmoc-L-Ala-OCH2PhOCH2CH2CO2H, TentaGel HL-NH2
resin instead of aminomethylated PS resin and using only automated SPPS
(coupling time: 1 h) to assemble the peptide chain. Lyophilisation
yielded 145.7 mg of crude EEEEEEAKSpAKSpAK.

[0776] A portion of the crude peptide (21.9 mg) was purified using a
linear gradient of 0% to 40% B over 20 min instead of 0% to 51% B over 33
minutes. Lyophilisation yielded the purified peptide
EEEEEEAKSpAKSpAK (3.7 mg, 14%) as a white solid in ca. 98%
purity according to analytical HPLC. Rt 13.8 min (0-40% B over 15
min, 1 mL/min).

(ii) Phosphopeptides 304-306

[0777] Peptides 304-306 were prepared from crude peptides 300-302,
respectively, by the following general procedure.

[0778] Microwave-enhanced click reactions were performed in a sealed glass
reaction vessel on a CEM Discover 908010 microwave reactor with
IR-monitored temperature control.

[0786] Iron nanoparticles were synthesised by coprecipitation in the
presence of phosphopeptide 107. The results are provided below in Table
1.

[0787] In a typical experiment, analogue 107 was weighed into a 0.2 mL
vial, a solution of 0.7 mol.L-1NH3.H2O (31.5 mL, 22 mmol)
was added and the mixture stirred until dissolution was complete. A drop
of aqueous 1 mol.L-1FeCl3 (2.5 mL, 2.5 mmol) and a drop of 1
mol.L-1 FeSO4 in 1M HCl (1.25 mL, 1.25 mmol) were deposited on
the walls of the vial. The vial was then flushed with N2, fixed to a
vortex shaker and stirred at 1800 rpm to mix the iron salt solutions with
the ammonia solution. A black precipitate quickly appeared. After 30 min,
the nanoparticles were separated from the supernatant liquor by
centrifugation, washed twice with distilled water (under nitrogen
atmosphere), freeze-dried and stored under nitrogen atmosphere.

[0788] The particles prepared in the presence of phosphopeptide 107 were
significantly more stable in suspension and therefore more difficult to
separate by centrifugation, than the particles prepared in the absence of
additives, which were easily separated by centrifugation.

[0789] The particles prepared in the presence of phosphopeptide 107 showed
signs of oxidation after storage overnight, indicated by a change in the
colour of the black powder initially obtained to a red-brown powder. In
contrast, the particles prepared in the absence of phosphopeptides
remained black for several weeks.

[0790] Accurate determination of the size of the particles over a large
population was not possible, due to the highly aggregated nature of the
particles after evaporation of a droplet of a suspension of the particles
on the TEM grid. The particle sizes reported above correspond to
estimations of the average particle sizes, based on the sizes of
particles on the edges of the aggregates as determined using TEM.

[0791] Control experiments (Table 1, Entries a-d) were carried out to
confirm the reproducibility of the synthesis and its sensitivity to small
changes in the experimental protocol. The experimental procedures for
Entries a and b were identical and resulted in very similar iron oxide
nanoparticles with particle sizes of 10 nm (FIGS. 17 and 18) and 12 nm,
respectively. The particles displayed a tendency to aggregate and
generally showed spheroid morphology. The product nanoparticles were
obtained in the form of a black powder. Electron diffraction patterns
showed five diffuse rings that can be indexed to Fe3O4 (220),
Fe3O4 (311), Fe3O4 (400), Fe3O4 (511) and
Fe3O4 (440). EDS measurements confirmed the presence of iron
and oxygen.

[0792] Modification of the experimental procedure by varying parameters
such as the stirring speed (Table 1, Entry c) or the concentration of
ammonia in solution (Table 1, Entry d) did not significantly affect the
size, composition, or morphology of the nanoparticles.

[0793] The introduction of low ratios of phosphopeptide 107 (Table 1,
Entries e-g) as an additive resulted in a significant reduction in the
size of the product nanoparticles (FIGS. 19 and 20). The average diameter
of the iron oxide nanoparticles was reduced from about 10 nm to about 5
nm. In contrast to the nanoparticles obtained in the control experiments,
the nanoparticles obtained using phosphopeptide 107 were obtained in the
form of a powder that was initially black, but changed to red-brown
overnight. Electron diffraction patterns for these samples showed a
dramatic decrease in the crystallinity of the sample and therefore the
diffuse rings could not be precisely attributed. EDS measurements
confirmed the presence of iron. The particles prepared in the presence of
different ratios of phosphopeptide 107 to iron (Table 1, Entries e-g) had
relatively similar shape and similar size (within the variance
calculated).

[0794] The introduction of high ratios of phosphopeptide 107 (Table 1,
Entries h-j) as an additive resulted in no precipitation. Instead, the
colour of the reaction mixture slowly changed from yellow to orange over
about 5 minutes.

Preparation of Metal Nanoparticles by Reduction

Iron Nanoparticles

Using Phosphopeptides 107-111

[0795] Iron nanoparticles were prepared by reduction in the presence of
107-111.

[0796] FeSO4.7H2O (1.98 mg, 7 mmol) was dissolved in 2 mL of
previously degassed deionised water. Trisodium citrate (0.18 mg, 0.7
mmol) was added for Entry b (Table 2), phosphopeptide 107-111 and peptide
77 (0.35 mmol) were added for Entries c-h respectively, and
3-O-(phospho)-serine 112 (0.26 mg, 1.4 mmol) was added for Entry i. No
additive was used for Entry a. The mixtures were stirred until
dissolution was complete. NaBH4 (0.5 mg, 13 mmol) in 1 mL of
deionised water was then added and the mixture stirred vigorously under
an atmosphere of nitrogen. A black precipitate quickly appeared and the
mixture was further stirred for 10 min. The particles were magnetically
decanted, washed three times with ethanol, dried, and stored under
nitrogen atmosphere.

[0798] Reduction of iron (II) sulfate in water using NaBH4 in the
absence of any additives (Table 2, Entry a) produced iron-iron oxide
core-shell nanoparticles with an overall size of 58±13 nm and a shell
thickness of 3.2±1 nm (FIG. 1). The particles were attached to each
other in the form of chain-like structures that extended beyond a few
hundred nanometres. Due to their large size, the structures were not
stable when suspended in solution. The structures had a strong tendency
to aggregate and precipitated out of the solution after a few seconds.

[0799] Repeating the reduction in the presence of trisodium citrate (10:1
metal to citrate) produced particles with a size and morphology very
similar to those produced in Entry a (FIG. 2). The electron diffraction
pattern showed two diffuse rings that can be indexed to Fe (110) and Fe
(211). EDS measurements confirmed the presence of iron and oxygen in the
samples. The iron nanostructures prepared in the presence of sodium
citrate displayed weaker tendency to agglomerate and remained stable in
suspension for a longer period of time, than those prepared in the
absence of any additives.

[0800] The use of phosphopeptides 107-110 (Table 2, Entries c-f) as
additives in the synthesis of iron nanoparticles dramatically reduced the
particles size from about 60 nm to about 20 nm. The particles prepared in
the presence of these analogues were all similar in shape and size
(within the variance calculated). These iron-iron oxide core-shell
nanoparticles were also joined together as chain-like structures, but
with nanoparticles of smaller diameter than the structures formed in
Entries a and b (FIGS. 7-14). However, in contrast to the structures
formed in Entries a and b, the structures formed in the presence of
phosphopeptides 107-110 formed stable suspensions when dispersed in
ethanol. Electron diffraction patterns for these samples showed four
diffuse diffraction rings that can be indexed to Fe3O4 (311),
Fe (110), Fe (200) and Fe (211). EDS measurements confirmed the presence
of iron and oxygen.

[0801] The use of phosphopeptide 111 as additive reduced the size of the
resulting nanoparticles more significantly, resulting in highly
aggregated nanoparticles with an average diameter of 8.8±2 nm (FIGS.
15 and 16). The particles were not core-shell nanoparticles. In contrast
to the particles obtained in the control experiments, which were obtained
in the form of black powders that remained black for several days, the
particles prepared in presence of phosphopeptide 111 were obtained in the
form of a powder that was initially black, but changed to grey overnight.
The electron diffraction pattern showed two diffuse rings that can be
indexed to either Fe3O4 (311) and Fe3O4 (440) or to
γ-Fe2O3 (311) and γ-Fe2O3 (440). EDS
confirmed the presence of iron and oxygen.

[0802] Control experiments were also carried out with peptide 77 (Table 2,
Entry h) or 3-O-(phospho)-serine 112 (Table 2, Entry i). Carrying out the
experimental procedure using peptide 77 as the additive gave rise to
large iron-iron oxide core-shell nanoparticles with a diameter of 49±7
nm (FIGS. 5 and 6), while the use of 3-O-(phospho)-serine 112 led to the
formation of irregularly shaped iron-iron oxide core-shell nanoparticles
with an average diameter of 29±8 nm (FIGS. 3 and 4). These
nanoparticles also formed chain-like structures. The electron diffraction
patterns for these nanoparticles showed three diffuse rings that can be
indexed to Fe3O4 (311), Fe (110) and Fe (211). EDS confirmed
the presence of iron and oxygen.

[0803] For Entries a-g, h, and i (Table 2), the thickness of the iron
oxide shells was measured to be about 4 nm.

[0804] Diffuse diffraction rings shown by the electron diffraction
indicated that all of the samples had low crystallinity. This was
confirmed by HR-TEM of the samples, where no lattice fringes were
observed for any of the samples, highlighting the absence of long range
ordering of the iron atoms within each nanoparticle.

[0805] The magnetic properties of the nanoparticles obtained in the
presence of trisodium citrate and in the presence of phosphopeptide 107
(Table 2, Entries b and c) were evaluated. M(H) loop measurements were
obtained between ±60 kOe at both T=10 K and T=300 K. The masses of
sample used were very small therefore the magnetic moment observed could
not be related to the magnetization in emu.g-1.

[0806] The M(H) plot for the iron-iron oxide core-shell nanoparticles
grown in presence of trisodium citrate (FIG. 21) shows a clear
ferromagnetic behaviour of the particles with the observation of a
magnetic hysteresis at both 10 K and 300 K. At low fields, sudden steps
in the magnetic moment were observed with the magnetisation suddenly
dropping when decreasing field approaches 0 Oe and the magnetisation
suddenly increasing when increasing field approaches 0 Oe. The size of
the steps decreased when the sample was measured at 300 K.

[0807] The M(H) plot for the iron nanoparticles grown in the presence of
analogue 107 also shows a clear ferromagnetic behaviour of the particles
with observation of a magnetic hysteresis at both 10 K and 300 K (FIG.
22). Steps in the magnetic moment around low field (similar to the ones
observed in FIG. 21) can be clearly observed at T=10 K. Again, the size
of these steps decreases with increasing temperature, until they
disappear at T=300 K. These steps affected the value of the sample
coercivity and remanent magnetization as a function of temperature,
inducing a dramatic increase in remanent magnetization at low
temperature. A small shift of the magnetic hysteresis loop in the
direction of the applied field was also observed at low temperature, but
disappeared above 25 K. The amplitude of this shift was similar whether
the system was cooled at zero fields or with an applied field of 6 T, the
latter being known to sometimes increase the exchange bias phenomenon.
The saturation moment decreases with increasing temperature.

Using Phosphopeptides 207, 209, 211, and 303-306

[0808] Iron nanoparticles were also prepared by reduction in the presence
of phosphopeptides 207, 209, 211, and 303-306.

[0809] FeSO4.7H2O (1.95 mg, 7 mmol) was dissolved in 1-1.5 mL of
previously degassed distilled water and the phosphopeptide (0.35 mmol)
was dissolved in 1 mL of previously degassed distilled water. The
solutions were mixed and stirred under nitrogen for 15 min. Then the
NaBH4 solution (40 mmol, 20 μL of a 2M solution in triethylene
glycol dimethyl ether) was added all at once and the reaction solution
stirred vigorously under nitrogen atmosphere (stirring speed 1000 rpm). A
black precipitate immediately appeared and the mixture was stirred for 10
min. The reaction mixture was sonicated in order to separate all
particles from the stirrer bar, centrifuged and the supernatant solution
was decanted. The black precipitate was suspended in degassed ethanol,
sonicated for 5 min, centrifuged and the supernatant decanted (2×)
and then the particles were dried in vacuo.

[0810] The particles formed in the presence of phosphopeptides 207, 209,
and 211 were iron-iron oxide core-shell nanoparticles. The particles size
and shell thickness are provided below in Table 3.

[0811] The particles were spheroidal in shape and aggregated in chain-like
structures (FIGS. 23-28). The monodispersity was good.

[0812] The nanoparticles formed stable suspensions, for example a
suspension of the iron nanoparticles formed using 207 was stable
overnight.

[0813] Electron diffraction patterns for the particles showed four diffuse
diffraction rings that can be indexed to Fe3O4 (311), Fe (110),
Fe (200) and Fe (211). EDS measurements confirmed the presence of iron
and oxygen.

[0814] The particles formed in the presence of 303-306 are shown in FIGS.
29-32. The particle sizes and shell thicknesses of the particles are
provided below in Table 3. The particles formed in the presence of
phosphopeptide 304 were highly aggregated and the electron diffraction
pattern is very faint.

[0815] A control experiment without phosphopeptide was also carried out.
Iron-iron oxide core-shell nanoparticles were formed. The particle size
and shell thickness of the particles is provided in Table 3. The
particles displayed a high degree of polydispersity. The electron
diffraction pattern showed three diffuse rings which can be indexed to Fe
(110), Fe (200) and Fe (211). EDS measurements confirmed that the sample
had high iron and oxygen content. The particles have a strong tendency to
aggregate and precipitate out of the suspension in ethanol after only a
couple of minutes.

[0816] Dynamic light scattering experiments were carried out on
nanoparticles prepared in the presence of 209. 1 mL of the crude reaction
mix was diluted with 3 mL of NaHCO3/Na2CO3 buffer (pH=10)
and 0.1% (v/v) Triton X-100 (reduced form) was added to break up the
aggregates. Then the sample was sonicated at 45° C. for 20 min.
The theoretical material refractive index of magnetite (2.42) and
dispersant viscosity, refractive index and dielectric constant of pure
water were used to characterise the sample. The measurement was carried
out at 65° C. and revealed an average particle size of 10.0 nm
(FIG. 33).

[0817] The magnetic properties of nanoparticles prepared in the presence
of 209 were evaluated by measuring the magnetic hysteresis loop at 300 K
from -6 to 6 Tesla. The magnetisation curve intercepts at the origin,
indicating an absence of remnant magnetisation (MR) and coercivity
(HC) (FIG. 34). The nanoparticles exhibit superparamagnetic
behaviour.

[0818] Quantitative chemical analyses of nanoparticles prepared in the
presence of 209 were collected using an X-ray energy dispersive
spectroscopy attachment in the STEM mode (scanning TEM) at an electron
beam accelerating voltage of 200 kV. FIG. 35 shows the bright field image
of the scanned area (a) and the elemental maps recorded for Fe (b), 0
(c), Na (d), N (e) and P (f). The measurement confirms that the high iron
content seen in standard

[0819] EDS analysis is located in the area of the depicted nanoparticles
in the STEM image. The abundant presence of oxygen atoms stems from the
iron oxide shell of the core/shell particles as well as phosphopeptide
209, which is still on the surface of the particles. This is further
confirmed by a nitrogen content of circa 10% and a slight enrichment of
phosphorous in the analysed area. The sodium atoms were introduced during
the synthesis of the nanoparticles using NaBH4 as reducing agent and
are possibly bound to oxide anions of the iron oxide shell or carboxylate
anions of phosphopeptide 209.

Platinum Nanoparticles

[0820] Platinum nanoparticles were prepared by reduction in the presence
of phosphopeptide 209.

[0821] Pt(NH3)4(NO3)2 (1.36 mg, 3.5 mmol) was
dissolved in 1 mL of previously degassed distilled water and the
phosphopeptide (0.33 mg. 0.175 mmol) was dissolved in 1 mL of previously
degassed distilled water. The solutions were mixed and stirred under
nitrogen for 15 min (colourless solution). Then the NaBH4 solution
(35 mmol, 17.5 μL of a 2M solution in triethylene glycol dimethyl
ether) was added all at once and the reaction solution stirred vigorously
under nitrogen atmosphere (stirring speed 1000 rpm) for 2 h. The reaction
mixture turned dark during that time. The mixture was sonicated,
centrifuged and the supernatant solution was decanted. The black
precipitate was suspended in degassed ethanol (500 μL), sonicated for
5 min, centrifuged and the supernatant decanted (2×) and then the
particles were dried in vacuo.

[0822] A control experiment without 209 was also carried out.

[0823] Pt(NH3)4(NO3)2 (3.5 mmol) dissolved in 2 mL of
degassed distilled water, then 17.5 μl 2M NaBH4 solution in
triglyme (35 mmol) added. The reaction mixture was stirred for 15 min
under nitrogen, then sonicated, centrifuged and the supernatant solution
decanted. The precipitate was suspended in degassed ethanol (500 μL),
sonicated for 5 min, centrifuged and the supernatant decanted (2×)
and then the particles were dried in vacuo.

[0824] Reduction in the presence of 209 provided large, very polydisperse
aggregates with a diameter of 163±63 nm (FIGS. 36 and 37). The
electron diffraction pattern of the nanoparticles is shown in FIG. 38.
EDS confirmed that the nanoparticles have high Pt content.

Palladium Nanoparticles

[0825] Palladium nanoparticles were prepared by reduction in the presence
of phosphopeptide 209.

[0826] PdCl2 (0.62 mg, 3.5 mmol) was dissolved in 1 mL of previously
degassed distilled water and the phosphopeptide (0.33 mg. 0.175 mmol) was
dissolved in 1 mL of previously degassed distilled water. The solutions
were mixed and stirred under nitrogen for 15 min (brown solution). Then
the NaBH4 solution (35 mmol, 17.5 μL of a 2M solution in
triethylene glycol dimethyl ether) was added all at once and the reaction
solution stirred vigorously under nitrogen atmosphere (stirring speed
1000 rpm) for 15 min. The reaction mixture turned black immediately upon
addition of the reducing agent. The mixture was sonicated, centrifuged
and the supernatant solution was decanted. The black precipitate was
suspended in degassed ethanol (500 μL), sonicated for 5 min,
centrifuged and the supernatant decanted (2×) and then the
particles were dried in vacuo.

[0827] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using PdCl2 instead of
Pt(NH3)4(NO3)2.

[0828] Reduction in the presence of 209 provided nanowires having an
average diameter of 5.0±1.1 nm (FIGS. 39 and 40). Many crystal fringes
visible by HRTEM. The electron diffraction pattern of the nanoparticles
is shown in FIG. 41. EDS confirmed that the nanoparticles have high Pd
content.

Ruthenium Nanoparticles

[0829] Ruthenium nanoparticles were prepared by reduction in the presence
of phosphopeptide 209.

[0830] RuCl3.xH2O (0.73 mg, 3.5 mmol) was dissolved in 1 mL of
previously degassed distilled water and the phosphopeptide (0.33 mg.
0.175 mmol) was dissolved in 1 mL of previously degassed distilled water.
The solutions were mixed and stirred under nitrogen for 15 min (dark
brown solution). Then the NaBH4 solution (35 mmol, 17.5 μL of a
2M solution in triethylene glycol dimethyl ether) was added all at once
and the reaction solution turned yellow, blue and then black within a
couple of seconds. After stirring vigorously under nitrogen atmosphere
(stirring speed 1000 rpm) for 15 min, the reaction mixture was sonicated,
centrifuged and the supernatant solution was decanted. The black
precipitate was suspended in degassed ethanol (500 μL), sonicated for
5 min, centrifuged and the supernatant decanted (2×) and then the
particles were dried in vacuo.

[0831] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using RuCl3.xH2O instead of
Pt(NH3)4(NO3)2.

[0832] Reduction in the presence of 209 provided large and polydisperse
aggregated wires/sheets with an average diameter of 63±37 nm, which
extend over several micrometer nanowires (FIGS. 42, 43, and 44). No
crystal fringes are visible. The nanoparticles may be ruthenium-ruthenium
oxide core-shell nanoparticles. The electron diffraction pattern shows
very faint rings (FIG. 45). EDS confirmed that the nanoparticles have
high Ru content.

[0833] Quantitative chemical analyses of the nanoparticles were collected
using an X-ray energy dispersive spectroscopy (EDS) attachment in the
STEM mode (scanning TEM) at an electron beam accelerating voltage of 200
kV. FIG. 46 shows the bright field image of the scanned area (a) and the
elemental maps recorded for Ru (b), 0 (c), C (d), P (e) and Na (f). The
measurement confirms that the high ruthenium content seen in standard EDS
analysis is located in the area of the depicted nanoparticles in the STEM
image. The abundant presence of oxygen atoms may stem from the ruthenium
oxide shell of ruthenium-ruthenium oxide core/shell particles as well as
the phosphopeptide, which is still on the surface of the particles. This
is further confirmed by a high carbon and phosphorus content. The sodium
atoms were introduced during the synthesis of the nanoparticles using
NaBH4 as reducing agent and are possibly bound to oxide anions of
the ruthenium oxide shell or carboxylate anions of the phosphopeptide.

Silver Nanoparticles

[0834] Silver nanoparticles were prepared by reduction in the presence of
phosphopeptide 209.

[0835] Silver trifluoroacetate (0.77 mg, 3.5 μmol) was dissolved in 1
mL of previously degassed distilled water and the phosphopeptide (0.33
mg. 0.175 μmol) was dissolved in 1 mL of previously degassed distilled
water. The solutions were mixed and stirred under nitrogen for 15 min
(colourless solution). Then the NaBH4 solution (35 mmol, 17.5 μL
of a 2M solution in triethylene glycol dimethyl ether) was added all at
once and the reaction solution stirred vigorously under nitrogen
atmosphere (stirring speed 1000 rpm) for 15 min. The brown reaction
mixture was centrifuged and the supernatant solution decanted. The brown
precipitate was suspended in degassed ethanol (500 mL), sonicated for 5
min, centrifuged and the supernatant decanted (2×) and then the
brown-red particles were dried in vacuo.

[0836] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using silver trifluoroacetate instead of
Pt(NH3)4(NO3)2.

[0837] The suspension obtained in the control experiment was not stable in
EtOH after purification. In contrast, the nanoparticles prepared in the
presence of 209 were stable suspension for at least 8 days.

Rhodium Nanoparticles

[0838] Rhodium nanoparticles were prepared by reduction in the presence of
phosphopeptide 209.

[0839] RhCl3.xH2O (0.73 mg, 3.5 mmol) was dissolved in 1 mL of
previously degassed distilled water (orange solution) and the
phosphopeptide (0.33 mg. 0.175 mmol) was dissolved in 1 mL of previously
degassed distilled water. The solutions were mixed and stirred under
nitrogen for 15 min (yellow solution). Then the NaBH4 solution (35
mmol, 17.5 μL of a 2M solution in triethylene glycol dimethyl ether)
was added all at once and the reaction solution turned dark (black-brown)
immediately. The mixture was stirred vigorously under nitrogen atmosphere
(stirring speed 1000 rpm) for 15 min. The reaction mixture was
centrifuged and the supernatant solution decanted. The black precipitate
was suspended in degassed ethanol (500 mL), sonicated for 5 min,
centrifuged and the supernatant decanted (2×) and then the
particles were dried in vacuo.

[0840] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using RhCl3.xH2O instead of
Pt(NH3)4(NO3)2.

[0841] The crude rhodium reaction suspension in the control experiment was
not stable. Nanoparticles precipitated from the reaction mixture after
about 1 hour. In contrast, nanoparticles prepared using 209 were stable
in suspension for at least 7 days.

Gold Nanoparticles

[0842] Gold nanoparticles were prepared by reduction in the presence of
phosphopeptide 209.

[0843] AuCl3 (1.06 mg, 3.5 mmol) was dissolved in 1 mL of previously
degassed distilled water and the phosphopeptide (0.33 mg. 0.175 mmol) was
dissolved in 1 mL of previously degassed distilled water. The solutions
were mixed and stirred under nitrogen for 15 min (light yellow solution).
Then the NaBH4 solution (35 mmol, 17.5 μL of a 2M solution in
triethylene glycol dimethyl ether) was added all at once and the reaction
solution turned purple-black immediately. The mixture was stirred
vigorously under nitrogen atmosphere (stirring speed 1000 rpm) for 15
min. The reaction mixture was centrifuged and the supernatant solution
decanted. The dark purple precipitate was suspended in degassed ethanol
(500 μL), sonicated for 5 min, centrifuged and the supernatant
decanted (2×) and then the particles were dried in vacuo.

[0844] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using AuCl3 instead of
Pt(NH3)4(NO3)2.

[0845] In the control experiment, the particles aggregated and
precipitated during the reaction. In contrast, the nanoparticles prepared
using 209 were stable in suspension for at least 7 days.

[0846] Reduction in the presence of 209 provided very small, relatively
monodisperse nanoparticles with an average diameter of 4.4±0.7 nm
(FIGS. 47, 48, and 49). The nanoparticles are very crystalline--many
crystal fringes are visible. The electron diffraction pattern of the
nanoparticles is shown in FIG. 50. EDS confirmed that the nanoparticles
have high Au content. EDS also confirmed the presence of phosphorus,
which is present in 209.

Cobalt Nanoparticles

[0847] Cobalt nanoparticles were prepared by reduction in the presence of
phosphopeptide 209.

[0848] CoCl3. 6 H2O (0.83 mg, 3.5 mmol) was dissolved in 1 mL of
previously degassed distilled water and the phosphopeptide (0.33 mg.
0.175 mmol) was dissolved in 1 mL of previously degassed distilled water.
The solutions were mixed and stirred under nitrogen for 15 min
(colourless solution). Then the NaBH4 solution (35 mmol, 17.5 μL
of a 2M solution in triethylene glycol dimethyl ether) was added all at
once and the reaction solution turned dark brown immediately. The mixture
was stirred vigorously under nitrogen atmosphere (stirring speed 1000
rpm) for 15 min. The reaction mixture was sonicated, centrifuged and the
supernatant solution decanted. The black precipitate was suspended in
degassed ethanol (500 μL), sonicated for 5 min, centrifuged and the
supernatant decanted (2×) and then the particles were dried in
vacuo.

[0849] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using CoCl3. 6 H2O instead of
Pt(NH3)4(NO3)2.

[0850] The suspension obtained in the control experiment precipitated
after 2 hours.

[0851] The suspension obtained by reduction in the presence of 209 began
to precipitate after 4 hours, but the rate of preciptation was very slow.
Precipitation was complete after 6 days.

Nickel Nanoparticles

[0852] Nickel nanoparticles were prepared by reduction in the presence of
phosphopeptide 209.

[0853] Ni(OAc)2. 4 H2O (0.87 mg, 3.5 μmol) was dissolved in 1
mL of previously degassed distilled water and the phosphopeptide (0.33
mg. 0.175 μmol) was dissolved in 1 mL of previously degassed distilled
water. The solutions were mixed and stirred under nitrogen for 15 min
(colourless solution). Then the NaBH4 solution (35 μmol, 17.5
μL of a 2M solution in triethylene glycol dimethyl ether) was added
all at once and the reaction solution turned dark brown immediately. It
was stirred vigorously under nitrogen atmosphere (stirring speed 1000
rpm) for 15 min. The reaction solution was centrifuged at 14500 rpm for
10 min, but no precipitation occurred.

[0854] A control experiment without 209 was also carried out using a
procedure analogous to that described above for the platinum
nanoparticles, using Ni(OAc)2. 4 H2O instead of
Pt(NH3)4(NO3)2.

[0855] Black particles aggregated and precipitated during the control
experiment.

[0856] In contrast, reduction in the presence of 209 provided a brown
solution with only precipitated and changed colour after 6 days.

[0858] The metal nanoparticles and metal nanoparticle-phosphopeptide
complexes of the present invention have numerous applications, as would
be appreciated by a person skilled in the art. For example, the particles
may be used in cancer treatment by hyperthermia, contrast enhancement in
medical imaging, new drug delivery methods, and as catalysts.

[0859] Although the invention has been described by way of example and
with reference to particular embodiments, it is to be understood that
modifications and/or improvements may be made without departing from the
scope of the invention.